Cardiomyocyte Turnover Research Articles

Mouse Strain Determines Cardiac Growth Potential

RationaleThe extent of heart disease varies from person to person, suggesting that genetic background is important in pathology. Genetic background is also important when selecting appropriate mouse models to study heart disease. This study examines heart growth as a function of strain, specifically C57BL/6 and DBA/2 mouse strains.ObjectiveIn this study, we test the hypothesis that two strains of mice, C57BL/6 and DBA/2, will produce varying degrees of heart growth in both physiological and pathological settings.Methods and ResultsDifferences in heart dimensions are detectable by echocardiography at 8 weeks of age. Percentages of cardiac progenitor cells (c-kit+ cells) and mononucleated cells were found to be in a higher percentage in DBA/2 mice, and more tri- and quad-nucleated cells were in C57BL/6 mice. Cardiomyocyte turnover shows no significant changes in mitotic activity, however, there is more apoptotic activity in DBA/2 mice. Cardiomyocyte cell size increased with age, but increased more in DBA/2 mice, although percentages of nucleated cells remained the same in both strains. Two-week isoproterenol stimulation showed an increase in heart growth in DBA/2 mice, both at cardiomyocyte and whole heart level. In isoproterenol-treated DBA/2 mice, there was also a greater expression level of the hypertrophy marker, ANF, compared to C57BL/6 mice.ConclusionWe conclude that the DBA/2 mouse strain has a more immature cardiac phenotype, which correlates to a cardiac protective response to hypertrophy in both physiological and pathological stimulations.

Abstract 121: Cellular Origins of Postnatal Cardiomyogenesis in the Normal, Infarcted and Cell-Treated Heart

Background: Controversy surrounds the putative mechanisms of cardiomyocyte (CM) replenishment in adult mammals. Using genetic fate mapping and bone marrow allotransplantation, we investigated the postnatal origins of CMs in the normal, infarcted and cell-treated heart. Methods & Results: Tamoxifen pulsing of non-infarcted bitransgenic MerCreMer-ZEG mice (αMHC promoter driving inducible Cre) resulted in specific labeling of CMs by GFP. Comparison of BrdU incorporation (after 5 weeks of BrdU administration) in GFP+ and GFP- CMs (by flow cytometry, immunocytochemistry and histology) indicated that in the normal adult mouse heart, CM turnover occurs predominantly through proliferation of resident small mononucleated CMs at an annual rate of ~1.3%, independent from CM polyploidization, multinucleation, cell fusion or DNA repair. After myocardial infarction (MI), new CM formation in the border zone arises from both progenitor cells (PCs) as well as pre-existing CMs. Transplantation of cardiosphere-derived cells (CDCs) upregulates both host CM cycling and recruitment of PCs. To investigate the identity of endogenous PCs, bitransgenic mice underwent MI, followed by tamoxifen pulsing. One week later, FACS of myocyte-depleted hearts resulted in isolation of small non-myocyte GFP+ cells, which exhibit activated αMHC post-MI (~1.5% of cells in risk area vs <0.2% in sham controls). These novel PCs (Sca1+ [53%]/ nestin+ [11%]/ ckit-/ ABCG2-) express cardiac transcription factors (Nkx 2.5, GATA4, MEF2C, FOXC2), cardiogenic molecules (BMP2) and cardiac structural proteins (at both transcript & protein level), beat spontaneously in vitro and form CMs when injected into recipient hearts. Bone marrow transplantation demonstrated that recruited PCs do not arise from hematogenous seeding. Cell therapy with CDCs stimulates recruitment of PCs (~4.2% of cells in risk area), while improving heart function and increasing viable myocardium. Conclusions: The normal adult mouse heart replenishes lost CMs through proliferation of pre-existing CMs. After MI, proliferation of resident CMs increases, but new CMs also arise from recruited PCs. CDCs boost both CM proliferation as well as recruitment of endogenous PCs, resulting in myocardial regeneration.

Cardiomyocyte proliferation and progenitor cell recruitment underlie therapeutic regeneration after myocardial infarction in the adult mouse heart

Cardiosphere-derived cells (CDCs) have been shown to regenerate infarcted myocardium in patients after myocardial infarction (MI). However, whether the cells of the newly formed myocardium originate from the proliferation of adult cardiomyocytes or from the differentiation of endogenous stem cells remains unknown. Using genetic fate mapping to mark resident myocytes in combination with long-term BrdU pulsing, we investigated the origins of postnatal cardiomyogenesis in the normal, infarcted and cell-treated adult mammalian heart. In the normal mouse heart, cardiomyocyte turnover occurs predominantly through proliferation of resident cardiomyocytes at a rate of ∼1.3–4%/year. After MI, new cardiomyocytes arise from both progenitors as well as pre-existing cardiomyocytes. Transplantation of CDCs upregulates host cardiomyocyte cycling and recruitment of endogenous progenitors, while boosting heart function and increasing viable myocardium. The observed phenomena cannot be explained by cardiomyocyte polyploidization, bi/multinucleation, cell fusion or DNA repair. Thus, CDCs induce myocardial regeneration by differentially upregulating two mechanisms of endogenous cell proliferation.

From cell phenotype to epigenetic mechanisms: new insights into regenerating myocardium

The self-regenerating property of the adult myocardium is not a new discovery. Even though we could not confirm that the adult myocardium is a post-mitotic tissue, we should consider that its plasticity is extremely low. Studies are still in progress to decipher the mechanisms underlying the abovementioned potential fetal features of the adult heart. The modest results of several clinical trials based on the transplantation of millions of autologous stem cells into the dysfunctional heart have confirmed that the cross-talk of different signals, such as the microenvironment, promotes the regeneration of adult myocardium. Recent scientific evidence has revealed that cellular cross-talk does not depend on the action of a single cell phenotype. It is conceivable that the limited turnover of cardiomyocytes is ensured by the interplay of adult cardiac cells in response to environmental changes. The epigenetic state of a cell serves as a dynamic interface between the environment and phenotype. The epigenetic modulation of the adult cardiac cells by natural active compounds encourages further studies to improve myocardial plasticity. In this review, we will highlight the most relevant studies demonstrating the epigenetic modulation of myocardial regeneration without the use of stem cell transplantation.

Cardiomyocyte proliferation contributes to heart growth in young humans

The human heart is believed to grow by enlargement but not proliferation of cardiomyocytes (heart muscle cells) during postnatal development. However, recent studies have shown that cardiomyocyte proliferation is a mechanism of cardiac growth and regeneration in animals. Combined with evidence for cardiomyocyte turnover in adult humans, this suggests that cardiomyocyte proliferation may play an unrecognized role during the period of developmental heart growth between birth and adolescence. We tested this hypothesis by examining the cellular growth mechanisms of the left ventricle on a set of healthy hearts from humans aged 0-59 y (n = 36). The percentages of cardiomyocytes in mitosis and cytokinesis were highest in infants, decreasing to low levels by 20 y. Although cardiomyocyte mitosis was detectable throughout life, cardiomyocyte cytokinesis was not evident after 20 y. Between the first year and 20 y of life, the number of cardiomyocytes in the left ventricle increased 3.4-fold, which was consistent with our predictions based on measured cardiomyocyte cell cycle activity. Our findings show that cardiomyocyte proliferation contributes to developmental heart growth in young humans. This suggests that children and adolescents may be able to regenerate myocardium, that abnormal cardiomyocyte proliferation may be involved in myocardial diseases that affect this population, and that these diseases might be treatable through stimulation of cardiomyocyte proliferation.

Application of Biotechnology in Myocardial Regeneration-Tissue Engineering Triad: Cells, Scaffolds, and Signaling Molecules

Application of Biotechnology in Myocardial Regeneration-Tissue Engineering Triad: Cells, Scaffolds, and Signaling Molecules

Hybrid Mathematical Model of Cardiomyocyte Turnover in the Adult Human Heart

RationaleThe capacity for cardiomyocyte regeneration in the healthy adult human heart is fundamentally relevant for both myocardial homeostasis and cardiomyopathy therapeutics. However, estimates of cardiomyocyte turnover rates conflict greatly, with a study employing C14 pulse-chase methodology concluding 1% annual turnover in youth declining to 0.5% with aging and another using cell population dynamics indicating substantial, age-increasing turnover (4% increasing to 20%).ObjectiveCreate a hybrid mathematical model to critically examine rates of cardiomyocyte turnover derived from alternative methodologies.Methods and ResultsExamined in isolation, the cell population analysis exhibited severe sensitivity to a stem cell expansion exponent (20% variation causing 2-fold turnover change) and apoptosis rate. Similarly, the pulse-chase model was acutely sensitive to assumptions of instantaneous incorporation of atmospheric C14 into the body (4-fold impact on turnover in young subjects) while numerical restrictions precluded otherwise viable solutions. Incorporating considerations of primary variable sensitivity and controversial model assumptions, an unbiased numerical solver identified a scenario of significant, age-increasing turnover (4–6% increasing to 15–22% with age) that was compatible with data from both studies, provided that successive generations of cardiomyocytes experienced higher attrition rates than predecessors.ConclusionsAssignment of histologically-observed stem/progenitor cells into discrete regenerative phenotypes in the cell population model strongly influenced turnover dynamics without being directly testable. Alternatively, C14 trafficking assumptions and restrictive models in the pulse-chase model artificially eliminated high-turnover solutions. Nevertheless, discrepancies among recent cell turnover estimates can be explained and reconciled. The hybrid mathematical model provided herein permits further examination of these and forthcoming datasets.

On the Fabric of the Human Body

With the publication of De Humani Corporis Fabrica in 1543, Andreas Vesalius bestowed some of the greatest advancements of anatomic understanding since the time of Galen, correcting major misconceptions, eg, the notion that the great vessels originated in the liver. It took nearly 1500 years for this evolution in anatomic thinking to transpire. It is simultaneously humbling and invigorating to note that nearly 500 years later the study of anatomy is just as lively and full of evolutionary change. In this issue of Circulation , Kajstura and colleagues1 definitively dispatch the dogma of the heart as a terminally differentiated organ, a concept formulated a mere 40 years earlier, and in so doing completely recalibrate our understanding of cellular homeostasis in the healthy and diseased heart. Article see p 1869 In 1973, Radovan Zak2 of the University of Chicago stated unequivocally, “The adult heart enlarges only by enlargement of its component muscle cells. Division of ventricular muscle cells in mammals is not activated after cardiac injury.” This conclusion was largely consistent with work by Linzbach3 in 1960; however, in this earlier comprehensive histological analysis, there was a suggestion that under certain circumstances the number of cardiac muscle fibers might increase. The Anversa laboratory began suggesting the possibility of cardiomyocyte proliferation in 1980,4 and by 1992, the group began to document clear evidence for replication of cardiac muscle cells in response to physiological stress and myocardial injury in both experimental models5–8 and humans.9,10 These early works included such bold statements as “the recognition of factors controlling myocyte proliferation may provide a new approach for regeneration of damaged and lost myocardium, a phenomenon considered impossible for several decades.” Although some recognized the power and implications of these observations, the Pfeffers' call for open-mindedness …

Induction of Native Cardiomyocyte Cell Cycle Re-Entry by Transplanted Cells as a Novel Mechanism of Therapeutic Regeneration in the Heart

BackgroundThe extent of cardiomyocyte (CM) turnover in adult mammals remains unclear, while controversy surrounds the putative mechanisms of CM replenishment.HypothesisUsing a genetic fate mapping approach and objective methods of quantification, we investigated the ability of resident adult CMs to re-enter the cell cycle in the normal, infarcted and cell-treated heart.Methods & ResultsTamoxifen pulsing of bitransgenic MerCreMer-ZEG mice (aMHC promoter driving tamoxifen-inducible Cre), resulted in efficient (∼80%) and specific labeling of CMs by GFP. Mice were then subjected to: a) sham surgery, b) MI, and c) MI followed by cardiosphere-derived cell (CDC) injection. Mice were pulsed with BrdU daily for up to 5 weeks. CMs, isolated by Langendorff enzymatic dissociation, underwent FACS-sorting for GFP (∼98.9% GFP+, with minimal contamination by non-CMs) and subsequent flow cytometric analysis for BrdU and Ki67. The normal adult mouse heart contains a small pool of cycling resident CMs (∼0.08%/week, projecting to a CM turnover of ∼1.3-4%/yr), which roughly doubles during the first 3 weeks after MI. Transplantation of CDCs further dramatically upregulates the percentage of cycling host CMs (to ∼0.7%/week) (Fig 1), while boosting heart function and increasing viable myocardium. Immunocytochemistry of isolated CMs confirmed the flow cytometry results, while PCR microarray analysis showed upregulation of several genes associated with cell-cycle progression in resident CMs post MI and CDC therapy. Cycling CMs are smaller, more mononucleated and primarily located in the border zone. The observed phenomena could not be explained by stem cell differentiation, CM polyploidization, bi/multinucleation, cell fusion or DNA repair. Quantitative estimates indicate that CM proliferation accounts for ∼18% of the salutary effect of CDC therapy.Conclusions BackgroundThe extent of cardiomyocyte (CM) turnover in adult mammals remains unclear, while controversy surrounds the putative mechanisms of CM replenishment. The extent of cardiomyocyte (CM) turnover in adult mammals remains unclear, while controversy surrounds the putative mechanisms of CM replenishment. HypothesisUsing a genetic fate mapping approach and objective methods of quantification, we investigated the ability of resident adult CMs to re-enter the cell cycle in the normal, infarcted and cell-treated heart. Using a genetic fate mapping approach and objective methods of quantification, we investigated the ability of resident adult CMs to re-enter the cell cycle in the normal, infarcted and cell-treated heart. Methods & ResultsTamoxifen pulsing of bitransgenic MerCreMer-ZEG mice (aMHC promoter driving tamoxifen-inducible Cre), resulted in efficient (∼80%) and specific labeling of CMs by GFP. Mice were then subjected to: a) sham surgery, b) MI, and c) MI followed by cardiosphere-derived cell (CDC) injection. Mice were pulsed with BrdU daily for up to 5 weeks. CMs, isolated by Langendorff enzymatic dissociation, underwent FACS-sorting for GFP (∼98.9% GFP+, with minimal contamination by non-CMs) and subsequent flow cytometric analysis for BrdU and Ki67. The normal adult mouse heart contains a small pool of cycling resident CMs (∼0.08%/week, projecting to a CM turnover of ∼1.3-4%/yr), which roughly doubles during the first 3 weeks after MI. Transplantation of CDCs further dramatically upregulates the percentage of cycling host CMs (to ∼0.7%/week) (Fig 1), while boosting heart function and increasing viable myocardium. Immunocytochemistry of isolated CMs confirmed the flow cytometry results, while PCR microarray analysis showed upregulation of several genes associated with cell-cycle progression in resident CMs post MI and CDC therapy. Cycling CMs are smaller, more mononucleated and primarily located in the border zone. The observed phenomena could not be explained by stem cell differentiation, CM polyploidization, bi/multinucleation, cell fusion or DNA repair. Quantitative estimates indicate that CM proliferation accounts for ∼18% of the salutary effect of CDC therapy. Tamoxifen pulsing of bitransgenic MerCreMer-ZEG mice (aMHC promoter driving tamoxifen-inducible Cre), resulted in efficient (∼80%) and specific labeling of CMs by GFP. Mice were then subjected to: a) sham surgery, b) MI, and c) MI followed by cardiosphere-derived cell (CDC) injection. Mice were pulsed with BrdU daily for up to 5 weeks. CMs, isolated by Langendorff enzymatic dissociation, underwent FACS-sorting for GFP (∼98.9% GFP+, with minimal contamination by non-CMs) and subsequent flow cytometric analysis for BrdU and Ki67. The normal adult mouse heart contains a small pool of cycling resident CMs (∼0.08%/week, projecting to a CM turnover of ∼1.3-4%/yr), which roughly doubles during the first 3 weeks after MI. Transplantation of CDCs further dramatically upregulates the percentage of cycling host CMs (to ∼0.7%/week) (Fig 1), while boosting heart function and increasing viable myocardium. Immunocytochemistry of isolated CMs confirmed the flow cytometry results, while PCR microarray analysis showed upregulation of several genes associated with cell-cycle progression in resident CMs post MI and CDC therapy. Cycling CMs are smaller, more mononucleated and primarily located in the border zone. The observed phenomena could not be explained by stem cell differentiation, CM polyploidization, bi/multinucleation, cell fusion or DNA repair. Quantitative estimates indicate that CM proliferation accounts for ∼18% of the salutary effect of CDC therapy. Conclusions

To the heart of the problem. mIGF-1: local effort for global impact

To the heart of the problem. mIGF-1: local effort for global impact

Cardiac Side Population Cells

Over the past decade, extensive work in animal models and humans has identified the presence of adult cardiac progenitor cells, capable of cardiomyogenic differentiation and likely contributors to cardiomyocyte turnover during normal development and disease. Among cardiac progenitor cells, there is a distinct subpopulation, termed "side population" (SP) progenitor cells, identified by their unique ability to efflux DNA binding dyes through an ATP-binding cassette transporter. This review highlights the literature on the isolation, characterization, and functional relevance of cardiac SP cells. We review the initial discovery of cardiac SP cells in adult myocardium as well as their capacity for functional cardiomyogenic differentiation and role in cardiac regeneration after myocardial injury. Finally, we discuss recent advances in understanding the molecular regulators of cardiac SP cell proliferation and differentiation, as well as likely future areas of investigation required to realize the goal of effective cardiac regeneration.

The Hypoxic Epicardial and Subepicardial Microenvironment

Recent reports indicate that the adult mammalian heart is capable of limited, but measurable, cardiomyocyte turnover. While the lineage origin of the newly formed cardiomyocytes is not entirely understood, mounting evidence suggest that the epicardium and subepicardium may represent an important source of cardiac stem or progenitor cells. Stem cell niches are characterized by low oxygen tension, where stem cells preferentially utilize cytoplasmic glycolysis to meet their energy demands. However, it is unclear if the heart harbors similar hypoxic regions, or whether these regions house metabolically distinct cardiac progenitor populations. Here we identify the epicardium and subepicardium as the cardiac hypoxic niche based on [corrected] capillary density quantification, and localization of Hif-1α in the uninjured heart. We further demonstrate that this hypoxic microenvironment houses a metabolically distinct population of glycolytic progenitor cells. Finally, we show that Hif-1α regulates the glycolytic phenotype and progenitor properties of these cells. These findings highlight important anatomical and functional properties of the epicardial and subepicardial microenvironment, and the potential role of hypoxia signaling in regulation of cardiac progenitors.

Mitochondria as a source and target of lipid peroxidation products in healthy and diseased heart

The heart is a highly oxidative organ in which cardiomyocyte turnover is virtually absent, making it particularly vulnerable to accumulation of lipid peroxidation products (LPP) formed as a result of oxidative damage. Reactive oxygen and nitrogen species are the most common electrophiles formed during lipid peroxidation and lead to the formation of both stable and unstable LPP. Of the LPP formed, highly reactive aldehydes are a well-recognized causative factor in ageing and age-associated diseases, including cardiovascular disease and diabetes. Recent studies have identified that the mitochondria are both a primary source and target of LPP, with specific emphasis on aldehydes in cardiomyocytes and how these affect the electron transport system and Ca(2+) balance. Numerous studies have found that there are functional consequences in the heart following exposure to specific aldehydes (acrolein, trans-2-hexanal, 4-hydroxynonenal and acetaldehyde). Because these LPP are known to form in heart failure, cardiac ischaemia-reperfusion injury and diabetes, they may have an underappreciated role in the pathophysiology of these disease processes. Lipid peroxidation products are involved in the transcriptional regulation of endogenous anti-oxidant systems. Recent evidence demonstrates that transient increases in LPP may be beneficial in cardioprotection by contributing to mitohormesis (i.e. induction of anti-oxidant systems) in cardiomyocytes. Thus, exploitation of the cardioprotective actions of the LPP may represent a novel therapeutic strategy for future treatment of heart disease.

Cardiomyocyte Renewal in Humans

To the Editor: In a recent review article by Leri, Kajstura, and Anversa1 in Circulation Research , there are several misrepresentations and factual errors in the description of our study on birth dating of heart cells.2 These errors affect their conclusions, and it is thus important to make some clarifications. First, Leri et al1 claim that we analyzed 12 pathological hearts, which is incorrect. Briefly, only 1 of the 12 studied subjects had a history of cardiac disease (a previous myocardial infarction) and was the only individual who had heart enlargement or any medication for cardiovascular disease (nitrates). Another individual died of acute myocardial infarction and displayed myocardial hypertrophy and moderate coronary sclerosis at autopsy. Of the remaining 10 individuals, 4 had slight myocardial hypertrophy, slight coronary sclerosis, and/or slight fibrosis detected at autopsy, and 1 of these had hypertension. The remaining 6 of the 12 studied individuals had neither a history of cardiovascular disease nor any sign of cardiac pathology at autopsy. Detailed information, including all of the above, was provided in our original study (Table S2), and we addressed the potential role of cardiac pathology in some of the included cases in our original publication.2 Second, Leri et al1 claim that we assessed the birth date of a …

Abstract P036: Regional Characterization of Myocardial Renewal in Humans

We and other groups have reported cardiomyocyte renewal in adult human hearts. However, there is no consensus about magnitude and regional heterogeneity in myocardial turnover. Important requirements to quantify cellular turnover are the accurate identification of cardiomyocytes and ploidy level. We present a regional analysis of turnover in the adult myocardium, using the integration of atmospheric radiocarbon (14C) into genomic myocardial DNA. Cardiomyocyte nuclei were labeled with pericentriolar protein 1 (PCM-1) and isolated by flow cytometry. In order to improve estimates on cardiomyocyte turnover, nuclei were isolated according to their DNA content. A substantial fraction of cardiomyocyte nuclei in the right and left ventricle were polyploid: 28.9%±10.6% diploid, 58.9%±8.5% tetraploid and 12.1%±7.4% octaploid in the left ventricle and 47.9%±10.6% diploid, 46.6%±8.3% tetraploid and 5.6%±3.2% octaploid in the right ventricle. Cardiomyocyte turnover was analyzed according to their ploidy distribution in the left and right ventricle. Data from this ongoing study indicates a limited regenerative potential in both ventricles. A comprehensive mathematical analysis of regional turnover distribution will be presented. Non-cardiomyocytes were in average 18.0±2.3 years younger than the respective subjects, indicating a substantial turnover in the whole myocardium. The human heart has the capability to regenerate cardiomyocytes. However, the magnitude of this process does not allow for the renewal of the whole myocardium under homeostatic condition in a lifetime. Understanding the underlying mechanisms of cardiac renewal will open up new avenues to treat cardiac injuries.

Cardiac regeneration therapy: connections to cardiac physiology

Without heart transplantation, a large number of patients with failing hearts worldwide face poor outcomes. By means of cardiomyocyte regeneration, cardiac regeneration therapy is emerging with great promise as a means for restoring loss of cardiac function. However, the limited success of clinical trials using bone marrow-derived cells and myoblasts with heterogeneous constituents, transplanted at a wide range of cell doses, has led to disagreement on the efficacy of cell therapy. It is therefore essential to reevaluate the evidence for the efficacy of cell-based cardiac regeneration therapy, focusing on targets, materials, and methodologies. Meanwhile, the revolutionary innovation of cardiac regeneration therapy is sorely needed to help the millions of people who suffer heart failure from acquired loss of cardiomyocytes. Cardiac regeneration has been used only in limited species or as a developing process in the rodent heart; now, the possibility of cardiomyocyte turnover in the human heart is being revisited. In the pursuit of this concept, the use of cardiac stem/progenitor stem cells in the cardiac niche must be focused to usher in a second era of cardiac regeneration therapy for the severely injured heart. In addition, tissue engineering and cellular reprogramming will advance the next era of treatment that will enable current cell-based therapy to progress to "real" cardiac regeneration therapy. Although many barriers remain, the prevention of refractory heart failure through cardiac regeneration is now becoming a realistic possibility.

Building a New Heart From Old Parts

See related article, pages 1374–1386 The prevailing dogma in cardiac biology for almost a century has been that the adult mammalian heart is a terminally differentiated organ. According to this paradigm, a 100-year-old human heart is comprised of a population of cardiomyocytes that are as old as the individual. Over the last decade, work from the laboratory of Piero Anversa and others has challenged this dogma.1 Cardiac biologists are now confronted with an alternative conceptual framework proposing the heart as a dynamic, self-renewing organ, capable of cardiomyocyte turnover throughout life. Heart disease is the leading cause of death in the developed world. A major reason for this is that the adult mammalian heart has limited regenerative capacity following injury. The default healing response to myocyte loss in the adult mammalian heart (eg, following myocardial infarction) involves replacement of myocytes with fibrous, noncontractile scar tissue. The consequent loss of contractile units compromises cardiac function and ultimately leads to heart failure. Absence of regeneration in the adult mammalian heart contrasts with the impressive regenerative potential of lower vertebrates, such as urodele amphibians and teleost fish, which are capable of regenerating significant portions of ventricular myocardium following injury.2,3 Although studies in adult rodents and humans suggest that some myocyte repopulation occurs after myocardial infarction,4,5 this response is clearly limited and incapable of circumventing large-scale myocyte loss, fibrosis, and ultimately organ failure. Cardiomyocyte loss accompanies aging. The heart undergoes a series of structural and morphological changes during normal aging that are characterized by cardiomyocyte loss (through apoptosis, necrosis, and autophagy) and hypertrophy of remaining viable myocytes, as well as interstitial and perivascular fibrosis.6 Aging also renders the heart more susceptible to major cardiovascular events.6 Therefore, studies investigating the physiological turnover and aging of myocytes are …

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.

Differential induction of cellular proliferation, hypertrophy and apoptosis in H9c2 cardiomyocytes by exogenous tissue factor

Recent evidence has shown that prolonged exposure to exogenous tissue factor (TF) can alter the cellular functions of cardiomyocytes resulting in cardiac dysfunction. The effect of TF may arise from local inflammation within or in the vicinity of the heart. The aim of this study was to investigate the effect of TF on cardiomyocyte proliferation and growth. H9c2 rat cardiomyocytes were exposed to a range of concentrations of recombinant TF (rTF) (1.3-52ng/ml) for up to 10days and the outcome on cell proliferation and induction of apoptosis measured. At lower concentrations examined (1.3ng/ml), rTF had a proliferative influence on the H9c2 cells. In contrast, elevated concentrations of rTF (52ng/ml) induced cellular apoptosis as indicated by increased caspase-3 activity and nuclear localisation of p53. Moreover, incubation with intermediate concentrations of rTF (13ng/ml) resulted in an initial increase in proliferation but subsequently, led to cellular apoptosis by day 7 of the incubation. In order to determine if these effects induced hypertrophic cell growth, expression of mechano-growth factor (MGF) was analysed. Incubation of cells with rTF resulted in enhanced expression of MGF particularly at the intermediate concentrations of rTF (13ng/ml) as well as mean cellular transverse diameter. In addition, there was a rapid increase in the expression of atrial natriuretic factor (ANF) in the cells, on incubation with rTF but diminished rapidly when exposed to higher concentrations of rTF. These data indicate that exposure to increasing concentrations of rTF can accelerate the rate of cardiomyocyte turnover which may ultimately lead to depletion of viable cells within the heart. Moreover, at lower concentrations of rTF, the induction of cell proliferation together with hypertrophic markers indicates that rTF may contribute to the induction and progression of cardiac hypertrophy.

Human Cardiac Stem Cells

The view of the heart as a static organ implies that myocyte death and formation play a negligible role in cardiac homeostasis. Although stem cells have been unexpectedly identified in several organs, including the brain, kidney, lung, and skeletal muscle, the search for a cardiac stem cell (CSC) has been perceived as a futile effort, given the acknowledged lack of regenerative potential of the myocardium. Nevertheless, in the past several years, the demonstration of myocyte renewal in the normal and diseased heart has revealed a new, dynamic, and lively picture of this organ. Components of the cell cycle machinery and markers of cell replication have been detected in cardiomyocytes. The demonstration that karyokinesis and cytokinesis involve cells expressing contractile proteins has provided evidence that cardiomyocyte division occurs in the adult heart.1 More recently, pulse-chase assays with thymidine analogs,2 lineage tracing protocols,3 and 14C birth dating of cells4 have shown the existence of myocyte turnover, a process that has been found to differ in magnitude according to the methods used for its documentation and quantification. Article see p 2559 Over the years, the heart has provided us with the evidence that solves the critical problem of the origin of cardiomyocytes. At this moment in time, paraphrasing Eugenio Montale, we could say that the human heart is “on the verge of betraying [its] final secret offering the opportunity to uncover…the still point of the world, the link that won't hold, the thread to untangle that will finally lead to the heart of a truth.”5 Newly generated myocytes may derive from division of preexisting parenchymal cells or from activation and differentiation of resident CSCs. Discriminating between these two possibilities is not an easy task. Fate-mapping strategies, which are commonly used to track the origin of cells and …