In Vivo Quantitative Assessment of Myocardial Structure,Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).     

Analysis of mouse Rad54 expression and its implications for homologous recombination

Homologous recombination is one of the major pathways for repair of DNA double-strand breaks (DSBs). Important proteins in this pathway are Rad51 and Rad54. Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) that mediates pairing with and strand invasion of homologous duplex DNA with the assist of Rad54. We estimated that the nucleus of a mouse embryonic stem (ES) cells contains on average 4.7x10(5) Rad51 and 2.4x10(5) Rad54 molecules. Furthermore, we showed that the amount of Rad54 was subject to cell cycle regulation. We discuss our results with respect to two models that describe how Rad54 stimulates Rad51-mediated DNA strand invasion. The models differ in whether Rad54 functions locally or globally. In the first model, Rad54 acts in cis relative to the site of strand invasion. Rad54 coats the Rad51 nucleoprotein filament in stoichiometric amounts and binds to the target duplex DNA at the site that is homologous to the ssDNA in the Rad51 nucleoprotein filament. Subsequently, it promotes duplex DNA unwinding. In the second model, Rad54 acts in trans relative to the site of strand invasion. Rad54 binds duplex DNA distant from the site that will be unwound. Translocation of Rad54 along the duplex DNA increases superhelical stress thereby promoting duplex DNA unwinding.     

The response of mammalian cells to UV-light reveals Rad54-dependent and independent pathways of homologous recombination

Ultraviolet (UV) radiation-induced DNA lesions can be efficiently repaired by nucleotide excision repair (NER). However, NER is less effective during replication of UV-damaged chromosomes. In contrast, translesion DNA synthesis (TLS) and homologous recombination (HR) are capable of dealing with lesions in replicating DNA. The core HR protein in mammalian cells is the strand exchange protein RAD51, which is aided by numerous proteins, including RAD54. We used RAD54 as a cellular marker for HR to study the response of mammalian embryonic stem (ES) cells to UV irradiation. In contrast to yeast, ES cells lacking RAD54 are not UV sensitive. Here we show that the requirement for mammalian RAD54 is masked by active NER. By genetically inactivating NER and HR through disruption of the Xpa and Rad54 genes, respectively, we demonstrate the contribution of HR to chromosomal integrity upon UV irradiation. We demonstrate using chromosome fiber analysis at the individual replication fork level, that HR activity is important for the restart of DNA replication after induction of DNA damage by UV-light in NER-deficient cells. Furthermore, our data reveal RAD54-dependent and -independent contributions of HR to the cellular sensitivity to UV-light, and they uncover that RAD54 can compensate for the loss of TLS polymerase η with regard to UV-light sensitivity. In conclusion, we show that HR is important for the progression of UV-stalled replication forks in ES cells, and that protection of the fork is an interplay between HR and TLS.     

Analysis of ionizing radiation-induced foci of DNA damage repair proteins

Repair of DNA double-strand breaks by homologous recombination requires an extensive set of proteins. Among these proteins are Rad51 and Mre11, which are known to re-localize to sites of DNA damage into nuclear foci. Ionizing radiation-induced foci can be visualized by immuno-staining. Published data show a large variation in the number of foci-positive cells and number of foci per nucleus for specific DNA repair proteins. The experiments described here demonstrate that the time after induction of DNA damage influenced not only the number of foci-positive cells, but also the size of the individual foci. The dose of ionizing radiation influenced both the number of foci-positive cells and the number of foci per nucleus. Furthermore, ionizing radiation-induced foci formation depended on the cell cycle stage of the cells and the protein of interest that was investigated. Rad51 and Mre11 foci seemed to be mutually exclusive, though a small subset of cells did show co-localization of these proteins, which suggests a possible cooperation between the proteins at a specific moment during DNA repair.     

Ionizing radiation-induced foci formation of mammalian Rad51 and Rad54 depends on the Rad51 paralogs, but not on Rad52

Homologous recombination is of major importance for the prevention of genomic instability during chromosome duplication and repair of DNA damage, especially double-strand breaks. Biochemical experiments have revealed that during the process of homologous recombination the RAD52 group proteins, including Rad51, Rad52 and Rad54, are involved in an essential step: formation of a joint molecule between the broken DNA and the intact repair template. Accessory proteins for this reaction include the Rad51 paralogs and BRCA2. The significance of homologous recombination for the cell is underscored by the evolutionary conservation of the Rad51, Rad52 and Rad54 proteins from yeast to humans. Upon treatment of cells with ionizing radiation, the RAD52 group proteins accumulate at the sites of DNA damage into so-called foci. For the yeast Saccharomyces cerevisiae, foci formation of Rad51 and Rad54 is abrogated in the absence of Rad52, while Rad51 foci formation does occur in the absence of the Rad51 paralog Rad55. By contrast, we show here that in mammalian cells, Rad52 is not required for foci formation of Rad51 and Rad54. Furthermore, radiation-induced foci formation of Rad51 and Rad54 is impaired in all Rad51 paralog and BRCA2 mutant cell lines tested, while Rad52 foci formation is not influenced by a mutation in any of these recombination proteins. Despite their evolutionary conservation and biochemical similarities, S. cerevisiae and mammalian Rad52 appear to differentially contribute to the DNA-damage response.     

Dynamics of relative chromosome position during the cell cycle

The position of chromosomal neighborhoods in living cells was followed using three different methods for marking chromosomal domains occupying arbitrary locations in the nucleus; photobleaching of GFP-labeled histone H2B, local UV-marked DNA, and photobleaching of fluorescently labeled DNA. All methods revealed that global chromosomal organization can be reestablished through one cell division from mother to daughters. By simultaneously monitoring cell cycle stage in the cells in which relative chromosomal domain positions were tracked, we observed that chromosomal neighborhood organization is apparently lost in the early G1 phase of the cell cycle. However, the daughter cells eventually regain the general chromosomal organization pattern of their mothers, suggesting an active mechanism could be at play to reestablish chromosomal neighborhoods.