Kanar Alkass

Evidence for Cardiomyocyte Renewal in Humans

It 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.

Dynamics of Hippocampal Neurogenesis in Adult Humans

Adult-born hippocampal neurons are important for cognitive plasticity in rodents. There is evidence for hippocampal neurogenesis in adult humans, although whether its extent is sufficient to have functional significance has been questioned. We have assessed the generation of hippocampal cells in humans by measuring the concentration of nuclear-bomb-test-derived ¹⁴C in genomic DNA, and we present an integrated model of the cell turnover dynamics. We found that a large subpopulation of hippocampal neurons constituting one-third of the neurons is subject to exchange. In adult humans, 700 new neurons are added in each hippocampus per day, corresponding to an annual turnover of 1.75% of the neurons within the renewing fraction, with a modest decline during aging. We conclude that neurons are generated throughout adulthood and that the rates are comparable in middle-aged humans and mice, suggesting that adult hippocampal neurogenesis may contribute to human brain function.

Neurogenesis in the Striatum of the Adult Human Brain

In most mammals, neurons are added throughout life in the hippocampus and olfactory bulb. One area where neuroblasts that give rise to adult-born neurons are generated is the lateral ventricle wall of the brain. We show, using histological and carbon-14 dating approaches, that in adult humans new neurons integrate in the striatum, which is adjacent to this neurogenic niche. The neuronal turnover in the striatum appears restricted to interneurons, and postnatally generated striatal neurons are preferentially depleted in patients with Huntingtons disease. Our findings demonstrate a unique pattern of neurogenesis in the adult human brain.

The Age of Olfactory Bulb Neurons in Humans

Continuous turnover of neurons in the olfactory bulb is implicated in several key aspects of olfaction. There is a dramatic decline postnatally in the number of migratory neuroblasts en route to the olfactory bulb in humans, and it has been unclear to what extent the small number of neuroblasts at later stages contributes new neurons to the olfactory bulb. We have assessed the age of olfactory bulb neurons in humans by measuring the levels of nuclear bomb test-derived (14)C in genomic DNA. We report that (14)C concentrations correspond to the atmospheric levels at the time of birth of the individuals, establishing that there is very limited, if any, postnatal neurogenesis in the human olfactory bulb. This identifies a fundamental difference in the plasticity of the human brain compared to other mammals.

Dynamics of Cell Generation and Turnover in the Human Heart

The contribution of cell generation to physiological heart growth and maintenance in humans has been difficult to establish and has remained controversial. We report that the full complement of cardiomyocytes is established perinataly and remains stable over the human lifespan, whereas the numbers of both endothelial and mesenchymal cells increase substantially from birth to early adulthood. Analysis of the integration of nuclear bomb test-derived (14)C revealed a high turnover rate of endothelial cells throughout life (>15% per year) and more limited renewal of mesenchymal cells (<4% per year in adulthood). Cardiomyocyte exchange is highest in early childhood and decreases gradually throughout life to <1% per year in adulthood, with similar turnover rates in the major subdivisions of the myocardium. We provide an integrated model of cell generation and turnover in the human heart.

Dynamics of Oligodendrocyte Generation and Myelination in the Human Brain

The 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.

Prodynorphin CpG-SNPs associated with alcohol dependence: elevated methylation in the brain of human alcoholics

The genetic, epigenetic and environmental factors may influence the risk for neuropsychiatric disease through their effects on gene transcription. Mechanistically, these effects may be integrated through regulation of methylation of CpG dinucleotides overlapping with single‐nucleotide polymorphisms (SNPs) associated with a disorder. We addressed this hypothesis by analyzing methylation of prodynorphin (PDYN) CpG‐SNPs associated with alcohol dependence, in human alcoholics. Postmortem specimens of the dorsolateral prefrontal cortex (dl‐PFC) involved in cognitive control of addictive behavior were obtained from 14 alcohol‐dependent and 14 control subjects. Methylation was measured by pyrosequencing after bisulfite treatment of DNA. DNA binding proteins were analyzed by electromobility shift assay. Three PDYN CpG‐SNPs associated with alcoholism were found to be differently methylated in the human brain. In the dl‐PFC of alcoholics, methylation levels of the C, non‐risk variant of 3′‐untranslated region (3′‐UTR) SNP (rs2235749; C > T) were increased, and positively correlated with dynorphins. A DNA‐binding factor that differentially targeted the T, risk allele and methylated and unmethylated C allele of this SNP was identified in the brain. The findings suggest a causal link between alcoholism‐associated PDYN 3′‐UTR CpG‐SNP methylation, activation of PDYN transcription and vulnerability of individuals with the C, non‐risk allele(s) to develop alcohol dependence.

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

Assays to quantify myocardial renewal rely on the accurate identification of cardiomyocyte nuclei. We previously ¹⁴C 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 ¹⁴C 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.

Postmortem identification of hyperglycemia

The detection of diabetic coma postmortem requires accurate biochemical analysis. Due to continuous consumption of glucose by surviving cells postmortem, blood glucose levels decrease rapidly. Therefore, vitreous fluid has been used as a substitute in forensic practice, since it has a very low cell count. It has been repeatedly reported that the sum value of vitreous glucose and lactate should be used to estimate the original antemortem blood glucose level, based on the assumption that pre-existing glucose is gradually converted to lactate under anaerobic conditions during agonal phase and the early postmortem period. In this study, we applied a strategy including consistent sampling of vitreous fluid from the centre of both eyes of deceased subjects as soon as possible after arrival at the morgue, and immediate bedside analysis using a blood gas instrument. In total, 3076 cases were included during 2004-2006. We found that, after an initial drop of vitreous glucose during the very early postmortem period, the levels stayed stable for appreciable time postmortem. Analysis of a second sample collected at autopsy 1-3 days later gave similar results (R(2)=0.90). In contrast, the vitreous lactate levels showed a steady increase. This implies that the sum value of glucose and lactate increases with postmortem time, as reflected by vitreous potassium level. In fact, a statistically significant difference in the sum value was seen between subjects with potassium below 10 mmol/L (n=1086) and above 20 mmol/L (n=531), p<.001. In addition, in this large material, we did not identify a single case with circumstantial indication of hyperglycemia that only showed high vitreous lactate. We therefore suggest that vitreous glucose alone should be used to diagnose hyperglycemia postmortem and that the limit of 10 mmol/L should have a good specificity for diabetic coma, which theoretically would equal an original blood glucose value of about 26 mmol/L. As to the methodology, we found that sonication, centrifugation and addition of fluoride to the samples are unnecessary procedures when using a blood gas instrument. The strategy resulted in a doubling of the number of diabetic coma identified at our department compared to preceding period when analysis only was performed on selected cases.

No Evidence for Cardiomyocyte Number Expansion in Preadolescent Mice

The magnitude of cardiomyocyte generation in the adult heart has been heavily debated. A recent report suggests that during mouse preadolescence, cardiomyocyte proliferation leads to a 40% increase in the number of cardiomyocytes. Such an expansion would change our understanding of heart growth and have far-reaching implications for cardiac regeneration. Here, using design-based stereology, we found that cardiomyocyte proliferation accounted for 30% of postnatal DNA synthesis; however, we were unable to detect any changes in cardiomyocyte number after postnatal day 11. (15)N-thymidine and BrdU analyses provided no evidence for a proliferative peak in preadolescent mice. By contrast, cardiomyocyte multinucleation comprises 57% of postnatal DNA synthesis, followed by cardiomyocyte nuclear polyploidisation, contributing with 13% to DNA synthesis within the second and third postnatal weeks. We conclude that the majority of cardiomyocytes is set within the first postnatal week and that this event is followed by two waves of non-replicative DNA synthesis. This Matters Arising paper is in response to Naqvi et al. (2014), published in Cell. See also the associated Correspondence by Soonpaa et al. (2015), and the response by Naqvi et al. (2015), published in this issue.