Analysis of cardiac cell turnover in humans by radiocarbon dating and mathematical modeling

Cardiovascular disease is the largest cause of morbidity and mortality in the Western World. Disease progression often involves a loss of contracting cells, cardiomyocytes, which leads to cardiac failure and the need for heart transplantation with time. However the shortage of donor hearts is a large problem and a strong motivator for finding alternative solutions; this is the focus of regenerative heart medicine. For new treatment strategies to be effective we first need to better understand the potential and capacity of the heart and its cells. This thesis addresses two questions specifically: 1) Do cardiomyocytes renew in human hearts during healthy aging? 2) How does cardiac disease affect cardiomyocyte renewal? Studies in experimental animals and to a small extent in humans had previously not been able to resolve these questions, mainly because limitations in methods and ethical restrictions. We employed primarily two methodologies, 14C birth dating and mathematical modeling. 14C birth dating is a method developed within the Frisén group that exploits the changes in atmospheric 14C levels due to testing of nuclear weapons during the Cold War. The 14C concentration in the genomic DNA of a cell reflects when the cell was born, and hence the level of renewal. The core part of the mathematical model is a first order partial differential equation (PDE). It describes cells according to their age and how the distribution of ages changes as the individual grows older. We found that human cardiomyocytes in healthy hearts indeed renew throughout life, with a declining turnover not exceeding 1% per year in adult life, and that the cell number is established already at birth. Endothelial and mesenchymal cardiac cells are more dynamic, both in terms of changes in cell number and baseline turnover (Paper II and IV). Preliminary results indicate that ischemic heart disease and dilated cardiomyopathy can increase the renewal rate to 2.7% per year; however it is likely that individual turnover estimates differ from this, which may reflect the differences in disease etiology and patient specific manifestation (Paper I). In order to reach these conclusions we developed a method to isolate cardiomyocyte nuclei, based on the molecular markers, PCM-1, cTroponin T, and cTroponin I (Paper III). This work shows that adult cardiomyocytes in healthy and diseased hearts have a measurable regenerative capacity, suggesting that it can be exploited for developing new therapeutic strategies to treat heart disease

Dissociation of EphB2 Signaling Pathways Mediating Progenitor Cell Proliferation and Tumor Suppression

SummarySignaling proteins driving the proliferation of stem and progenitor cells are often encoded by proto-oncogenes. EphB receptors represent a rare exception; they promote cell proliferation in the intestinal epithelium and function as tumor suppressors by controlling cell migration and inhibiting invasive growth. We show that cell migration and proliferation are controlled independently by the receptor EphB2. EphB2 regulated cell positioning is kinase-independent and mediated via phosphatidylinositol 3-kinase, whereas EphB2 tyrosine kinase activity regulates cell proliferation through an Abl-cyclin D1 pathway. Cyclin D1 regulation becomes uncoupled from EphB signaling during the progression from adenoma to colon carcinoma in humans, allowing continued proliferation with invasive growth. The dissociation of EphB2 signaling pathways enables the selective inhibition of the mitogenic effect without affecting the tumor suppressor function and identifies a pharmacological strategy to suppress adenoma growth

Cardiomyocyte renewal in humans.

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Identification of cardiomyocyte nuclei and assessment of ploidy for the analysis of cell turnover.

International audienceAssays to quantify myocardial renewal rely on the accurate identification of cardiomyocyte nuclei. We previously (14)C birth dated human cardiomyocytes based on the nuclear localization of cTroponins T and I. A recent report by Kajstura et al. suggested that cTroponin I is only localized to the nucleus in a senescent subpopulation of cardiomyocytes, implying that (14)C birth dating of cTroponin T and I positive cell populations underestimates cardiomyocyte renewal in humans. We show here that the isolation of cell nuclei from the heart by flow cytometry with antibodies against cardiac Troponins T and I, as well as pericentriolar material 1 (PCM-1), allows for isolation of close to all cardiomyocyte nuclei, based on ploidy and marker expression. We also present a reassessment of cardiomyocyte ploidy, which has important implications for the analysis of cell turnover, and iododeoxyuridine (IdU) incorporation data. These data provide the foundation for reliable analysis of cardiomyocyte turnover in humans

Dynamics of oligodendrocyte generation and myelination in the human brain.

International audienceThe myelination of axons by oligodendrocytes has been suggested to be modulated by experience, which could mediate neural plasticity by optimizing the performance of the circuitry. We have assessed the dynamics of oligodendrocyte generation and myelination in the human brain. The number of oligodendrocytes in the corpus callosum is established in childhood and remains stable after that. Analysis of the integration of nuclear bomb test-derived (14)C revealed that myelin is exchanged at a high rate, whereas the oligodendrocyte population in white matter is remarkably stable in humans, with an annual exchange of 1/300 oligodendrocytes. We conclude that oligodendrocyte turnover contributes minimally to myelin modulation in human white matter and that this instead may be carried out by mature oligodendrocytes, which may facilitate rapid neural plasticity

Evidence for cardiomyocyte renewal in humans.

International audienceIt has been difficult to establish whether we are limited to the heart muscle cells we are born with or if cardiomyocytes are generated also later in life. We have taken advantage of the integration of carbon-14, generated by nuclear bomb tests during the Cold War, into DNA to establish the age of cardiomyocytes in humans. We report that cardiomyocytes renew, with a gradual decrease from 1% turning over annually at the age of 25 to 0.45% at the age of 75. Fewer than 50% of cardiomyocytes are exchanged during a normal life span. The capacity to generate cardiomyocytes in the adult human heart suggests that it may be rational to work toward the development of therapeutic strategies aimed at stimulating this process in cardiac pathologies

The age and genomic integrity of neurons after cortical stroke in humans

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The age and genomic integrity of neurons after cortical stroke in humans.

International audienceIt has been unclear whether ischemic stroke induces neurogenesis or neuronal DNA rearrangements in the human neocortex. Using immunohistochemistry; transcriptome, genome and ploidy analyses; and determination of nuclear bomb test-derived (14)C concentration in neuronal DNA, we found neither to be the case. A large proportion of cortical neurons displayed DNA fragmentation and DNA repair a short time after stroke, whereas neurons at chronic stages after stroke showed DNA integrity, demonstrating the relevance of an intact genome for survival