Limitations of the MRL mouse as a model for cardiac regeneration.

OBJECTIVE: Myocardial repair following injury in mammals is restricted such that damaged areas are replaced by scar tissue, impairing cardiac function. MRL mice exhibit exceptional regenerative healing in an ear punch wound model. Some myocardial repair with restoration of heart function has also been reported following cryoinjury. Increased cardiomyocyte proliferation and a foetal liver stem cell population were implicated. We investigated molecular mechanisms facilitating myocardial repair in MRL mice to identify potential therapeutic targets in non-regenerative species. METHODS: Expressions of specific cell-cycle regulators that might account for regeneration (CDKs 1, 2, 4 and 6; cyclins A, E, D1 and B1; p21, p27 and E2F5) were compared by immunoblotting in MRL and control C57BL/6 ventricles during development. Flow cytometry was used to investigate stem cell populations in livers from foetal mice, and infarct sizes were compared in coronary artery-ligated and sham-treated MRL and C57BL/6 adult mice. KEY FINDINGS: No differences in the expressions of cell cycle regulators were observed between the two strains. Expressions of CD34+Sca1+ckit-, CD34+Sca1+ckit+ and CD34+Sca1-ckit+ increased in livers from C57BL/6 vs MRL mice. No differences were observed in infarct sizes, levels of fibrosis, Ki67 staining or cardiac function between MRL and C57BL/6 mice. CONCLUSIONS: No intrinsic differences were observed in cell cycle control molecules or stem cell populations between MRL and control C57BL mouse hearts. Pathophysiologically relevant ischaemic injury is not repaired more efficiently in MRL myocardium, questioning the use of the MRL mouse as a reliable model for cardiac regeneration in response to pathophysiologically relevant forms of injury.

Over expression of Plk1 does not induce cell division in rat cardiac myocytes in vitro.

BACKGROUND: Mammalian cardiac myocytes withdraw from the cell cycle during post-natal development, resulting in a non-proliferating, fully differentiated adult phenotype that is unable to repair damage to the myocardium, such as occurs following a myocardial infarction. We and others previously have shown that forced expression of certain cell cycle molecules in adult cardiac myocytes can promote cell cycle progression and division in these cells. The mitotic serine/threonine kinase, Polo-like kinase-1 (Plk1), is known to phosphorylate and activate a number of mitotic targets, including Cdc2/Cyclin B1, and to promote cell division. PRINCIPAL FINDINGS: The mammalian Plk family are all differentially regulated during the development of rat cardiac myocytes, with Plk1 showing the most dramatic decrease in both mRNA, protein and activity in the adult. We determined the potential of Plk1 to induce cell cycle progression and division in cultured rat cardiac myocytes. A persistent and progressive loss of Plk1 expression was observed during myocyte development that correlated with the withdrawal of adult rat cardiac myocytes from the cell cycle. Interestingly, when Plk1 was over-expressed in cardiac myocytes by adenovirus infection, it was not able to promote cell cycle progression, as determined by cell number and percent binucleation. CONCLUSIONS: We conclude that, in contrast to Cdc2/Cyclin B1 over-expression, the forced expression of Plk1 in adult cardiac myocytes is not sufficient to induce cell division and myocardial repair.

The use of proteomics to identify novel therapeutic targets for the treatment of disease.

The completion of the Human Genome Project has revealed a multitude of potential avenues for the identification of therapeutic targets. Extensive sequence information enables the identification of novel genes but does not facilitate a thorough understanding of how changes in gene expression control the molecular mechanisms underlying the development and regulation of a cell or the progression of disease. Proteomics encompasses the study of proteins expressed by a population of cells, and evaluates changes in protein expression, post-translational modifications, protein interactions, protein structure and splice variants, all of which are imperative for a complete understanding of protein function within the cell. From the outset, proteomics has been used to compare the protein profiles of cells in healthy and diseased states and as such can be used to identify proteins associated with disease development and progression. These candidate proteins might provide novel targets for new therapeutic agents or aid the development of assays for disease biomarkers. This review provides an overview of the current proteomic techniques available and focuses on their application in the search for novel therapeutic targets for the treatment of disease.

Sperm proteome mapping of a patient who experienced failed fertilization at IVF reveals altered expression of at least 20 proteins compared with fertile donors: case report.

The aim of this study was to compare the sperm protein expression profile (proteome map) from a patient who experienced failed fertilization at IVF with fertile controls. One patient and three fertile donor sperm samples were characterized using two-dimensional electrophoresis. Differences in protein expression were established using gel analysis software before attempted protein identification. Gel analysis of the fertile donor proteome maps revealed excellent reproducibility as well as very low intra-donor and inter-donor variability in the presence of protein spots. In the patient samples, we have noted 20 consistent differences in protein expression (six spots missing, three additional spots, four less abundant, seven more abundant) compared with the controls. Two proteins that were more intense in the patient have been conclusively identified as secretory actin-binding protein and outer dense fibre protein 2/2. In conclusion proteome variation between different fertile donors was very low. In contrast, the patient proteome exhibited 20 differences compared with controls, which we believe is an underestimate. These proteins merit further investigation to determine whether failed fertilization at IVF might be caused by abnormalities in their expression. This case report represents a proof of principle that proteomics may be useful to study defects in sperm function.

Physiological and proteomic approaches to studying prefertilization events in the human.

This research aims firstly to understand, in cellular and molecular terms, how a mature human spermatozoon is prepared for fertilization, and secondly, to identify what factors are involved in the initial signalling interactions between the egg and spermatozoon. In order to achieve these objectives, a combination of approaches is being used, including single-cell imaging, patch clamping and proteomics. Single-cell imaging reveals hidden complexity and heterogeneity in signalling responses in spermatozoa. Characterization of cell physiology at the single-cell level must be a future aim, including the study of ion channel expression and function by patch clamping. Proteomic experiments are aimed at identifying defects in protein expression in specific subgroups of men, e.g. those with globozoospermia. A better understanding of prefertilization events will allow the development of non-assisted reproductive therapy, drug-based treatments for male infertility.