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Cardiac Death and Regeneration

Final Report Summary - CADRE (Cardiac Death and Regeneration)

Cardiac muscle death, without equivalent cardiomyocyte replacement, results in deficiencies in pump function that characterize myocardial infarction and those chronic cardiomyopathies where apoptosis is present. Thus, heart failure may be viewed, in significant part, as a deficiency in cardiomyocyte regenerative capacity, a view that has prompted significant interest in stimulating cardiac muscle regeneration after damage. For this reason, understanding the molecular events of cardiac regeneration in heart disease is of fundamental importance and also immense translational potential.

In this project we proposed to study the roles of telomerase and of the telomere-capping protein, TRF2. Aim 1, Determine the properties of adult cardiac progenitor cells in mice that lack the RNA component of telomerase (TERC). Aim 2, Determine the properties of adult cardiac progenitor cells in mice that lack the catalytic component of telomerase (TERT). Aim 3, Determine the properties of adult cardiac muscle and adult cardiac progenitor cells that lack the telomere-capping protein TRF2. Aim 4, Test the prediction that forced expression of TERT and TRF2 can augment cardiac muscle engraftment in vivo and enhance the clonal derivation of adult cardiac progenitor cells in vitro, without adversely affecting the cells’ differentiation potential.

Using stem cell antigen-1, we previously identified telomerase-expressing cells in adult mouse myocardium, which have salutary properties for bona fide cardiac regeneration. Our studies of heart-derived Sca-1+ cardiac progenitor cells with a “side population” (SP) phenotype have helped us to develop a better understanding of their multi-lineage stem cell potential and likely involvement in the natural regenerative capacity of the heart. In addition to these two cellular markers, our group more recently has been able to identify platelet-derived growth factor receptor alpha (PDGFR-α) as an important cell surface feature that correlated even more precisely with the clonogenic capacity and cardiogenic transcription factor profile of the larger heterogeneous Sca-1+ population, than does the SP dye-efflux assay used previously.
Systematic evaluation of the capacity of Sca-1+ SP cells has also enabled us to prove that the clonogenicity of these cells increases if cultured at lower oxygen concentration than in the hyperoxic stress of ambient oxygen, resembling better the tissue niche. This discovery emphasizes the notion that clonogenicity is a operational definition that is contingent on experimental conditions and suggests the testable hypothesis that the cells purified and cultured in this way have an increase in telomerase expression. Using quantitative telomere-specific fluorescence in situ hybridization (FISH), we have observed that cloned Sca-1+ cells that are kept in lower oxygen concentration have significantly longer telomeres. Additional characterisation of this phenomenon is currently being conducted to elucidate the mechanisms for the reduced telomere attrition and the contributions of different mechanisms of telomere length maintenance.
To further our understanding of the role of telomere maintenance in these cardiac progenitor cell populations and to incorporate our new findings regarding the potential role of PDGFR-α as an improved prospective maker of clonogenicity, we have isolated and produced mouse heart-derived Sca-1+ PDGFR-α+ SP and non-SP cell clones, from telomerase reverse transcriptase (TERT) knock-out, heterozygous, and wild-type animals. Surprisingly, we observed that the absence of TERT in first generation (G1) and second generation (G2) knock-out mice does not impair the initial capacity of these cells to grow at single cell density. Whereas this feature of the cells is independent of TERT, the cell clones generated by these experiments are being characterised to understand if deletion of TERT impairs the later expansion of these cells, their propensity to undergo replicative senescence, and the susceptibility to apoptosis even in derivatives of G1 mice. Having successfully created this unique resource of G1 and G2 cardiac stem cell lines, we are also in the process of fully characterising the clones at the level of their telomere length, cardiogenic transcriptional signature, and telomere maintenance by the TERT-independent pathway, so-called alternative lengthening of telomeres (ALT) by homologous recombination. We are also in the process of characterising the susceptibility of these cell clones to undergo replicative senescence and apoptosis.
We are currently testing the clonogenicity of cardiac stem cells from animals in the third generation of TERT abrogation (G3), by which stage telomere dysfunction should be evident in this genetic background. In parallel, we will compare samples from the G1, G2, and G3 TERT knockout allowing us to quantify the enzymatic activity of telomerase in different heart- derived populations of CPCs as well as terminally differentiated cells. Molecular characterisation of these novel resources will help us to understand the role of telomere maintenance in multipotent clonogenic cardiac stem cells and its contribution to the cells’ growth capacity, plasticity, and resistance to stress.
Additionally, we have also successfully generated important tools for the study of the role of telomerase in cardiac progenitor cells. A key effort has been made in the generation of lentiviral vectors expressing mouse and human TERT, cloned into pLVX-IRES vectors that co-express fluorescent reporter protein, along with vectors for the mouse non-coding TERC RNA, cloned into a pLL3.8 derivative, driven by the U6 promoter and also containing a PGK-driven fluorescent protein cassette. These new tools will enable us to perform rescue experiments in in vivo and in vitro conditions.
In parallel to these studies of the TERT knock-out, c (for which no specific lineage marker presently exists). The information we are generating with these novel models will be of significant relevance, especially in the light of recent publications suggesting a contribution of pre-existing cardiomyocytes, as well as CPCs, to heart regeneration after injury.