|
Our research projects include:
- Asynchronous contractions in the adult rat cardiocyte [Top]
 -
This computer simulation in Matlab is an ongoing project and is intended to address, in order, (1) calcium fluxes in the single sarcomere, (2) calcium fluxes responsible for sarcomere-to-sarcomere coupling in the myofibril, and (3) calcium fluxes in the intact cardiocyte, addressing both axial and longitudinal mechanical coupling and calcium flux. Sections (1) and (2) can be accomplished on a PC and have virtually been completed, while (3) has not been tackled and will require a parallel computing solution. This model builds particularly upon the published work of Peskin (NYU), Stern (NIH) and Bers (Loyola U.).
- Microarray Analysis to Measure the Effects of the Antidiabetic Drug Rosiglitazone on Gene Expression in Neonatal Rat Cardiomyocytes[Top]
 -
The purpose of this study is to measure the effects of Rosiglitazone on gene expression over a time course in isolated cultured neonatal rat cardiomyocytes. The data gives results from the Illumina RatRef-12 Expression BeadChip technology. Here, we will analyze such expression data to: (1) identify differentially expressed genes, (2) map genes into functional biological categories, and (3) provide physiological interpretation of the gene lists derived.
• see data analysis results
- The analysis of the time course of calcium resequestration mechanisms in the adult mammalian cardiac cell [Top]
 -
This analysis requires separation of the contributions made by the two major regulatory systems in providing for the re-uptake of free calcium during relaxation in adult rat myocytes mediated primarily by two systems serving to return cytosolic free calcium to diastolic levels: (1) the SR calcium pump, SERCA (Atp2a2, Atp2a3 genes), responsible for an estimated 92% of calcium reuptake, and (2) the Na-Ca exchanger NCX, (Slc8a1, Slc8a2 genes), providing most of the remaining 8% contribution. This project involves first gathering accurate experimental data with a maximum signal-to-noise ratio (100 carefully synchronized calcium transients for a 10:1 S:N improvement). Pharmacological interventions will be used to modulate the two relaxation mechanisms experimentally, measuring resulting transient magnitude and time course changes. The use of RNAi (RNA interference) involves the plasmid introduction of double stranded RNA (dsRNA) to specifically target a gene's product, resulting in null or attenuated phenotypes. Rajanya Shah (MS, 2002) in this laboratory previously tried antisense RNA to interfere with SERCA gene activity, with mixed results. It is now known that it is the presence of dsRNA, formed from the annealing of sense and antisense strands present in in vitro RNA preps, that is responsible for producing the interfering activity.
- Development of a valid contractility assay for neonatal cardiocytes [Top]
 -
A method to quantify muscle contractility in neonatal cells has not been available to researchers using this preparation. This problem involves both experimental bench work with neonatal cardiocytes, installation of the image capture hardware, and development of the necessary software to yield contractility records similar to the adult myocyte shortening records now analyzed for time to peak and relaxation, velocities of shortening and relaxation, etc. Several strategies are being pursued: (1) image analysis of contracting myocytes to measure perimeter changes in the cells during pacing; (2) tracking disordered myofibrillar fragments caught in a user-drawn Area of Interest (AOI) window on the image to demonstrate the equivalence of cell boundary changes with sarcomere shortening measurements. (3) Possible comparison with cell tension changes, however this might be measured, may be attempted with a contact micro-force transducer.
- Comparison of the time course of calcium regulatory genes expressed in neonatal and adult cardiac myocytes [Top]
 -
Neonatal data from the microarray experiment using qPCR will be compared with new measurements on mRNA extracts from adult cardiocytes. This experiment represents a mini-developmental biology study, since some gene expression may be switched from neonatal to adult isoforms. Results from this experiment could pave the way for further transition in the lab from neonatal to adult preparations.
- Simulation of calcium fluxes in the single sarcomere and the single myofibril of a cardiocyte [Top]
 -
(1) visualization of propagation of an excitation wave along a myofibril in an asynchronously contracting myocyte: 2-D and 3-D pseudocolor Matlab animations first using a simplified model data set, then model data generated from Antonio Martinez’ project. (2) visualization of spread of calcium in a single sarcomere: 2-D and 3-D pseudocolor animations first using a simplified model data set, then model data generated in Antonio Martinez’ project
- Matlab simulation of whole myocyte shortening dynamics [Top]
 -
Matlab simulation of whole myocyte shortening dynamics in an asynchronously, spontaneously propagated (not synchronously, electrically paced) contraction of an adult myocyte. Perhaps a HPC project with about one hundred myofibrils (5,000 sarcomeres) contracting out of phase, contained by a deformable bounding sarcolemmal membrane, with volume constancy constraint. This model might also eventually be driven by data generated by Antonio Martinez’ project.
- Comparison of the time course of calcium regulatory gene expression in neonatal and adult cardiac myocytes [Top]
 -
Neonatal gene expression levels from the microarray experiment using the Western blot technique will be compared with new measurements on adult cardiocytes. This experiment represents a mini-developmental biology study and is complementary to the qPCR experiments. Which genes are turned on and off, and which are expressed by different isoforms, neonatal and adult? Experiments will be analyzed using the Eagle-Eye scanning densitometer.
- Variations in the calcium transient (amplitude rate of release, rate of resequestration) mapped along length of a cardiocyte [Top]
-
This experiment would utilize one of two techniques: (1) with equipment available in the laboratory, using a thin slit aperture to scan single myocytes in sub-micrometer increments using the stage piezoelectric nanopositioner; (2) with the confocal microscope in the COS Electron Microscopy Facility, PS-1. Measurements obtained will be compared with model data used by Jennifer Winn.
- Alterations in the calcium transient with selected experimental perturbations [Top]
 -
Alterations in the calcium transient with selected experimental perturbations (e.g. other interesting drugs or inotropic agents affecting calcium release or calcium uptake kinetics).
- A model of cardiocyte shortening [Top]
 -
A model of cardiocyte shortening based on an elastic, deformable sarcolemmal membrane, intermyofibrillar linkages, and sarcomere restoring forces (e.g. from titin) to account for observed relaxation dynamics in the cardiocyte. In the lab, this could involve careful measurement and curve fitting of relaxation data, and the use of selective extraction of structural proteins from myocytes that provide the restoring forces.
- Skeletal fiber isometric force dependence on the length-tension curve [Top]
 -
This project involves computer simulation and prediction of the force generation in an isolated skeletal muscle fiber, with experimental verification of the force (measured directly with a force transducer during tetanus or during chemically induced contracture) and 3-dimensional structural data obtained from optical sections of the fiber through a segment of its volume. Can a whole fiber isometric length-tension behavior be predicted from its 3-D structure? That is, can the isometric length-tension relation be predicted structurally? Some date for this project has been collected.
|
