INSIGHT & FUTURE TREND

MYOCYTE RENEWAL AND VENTRICULAR
REMODELLING

Abstract

   Remaining young at heart is a desirable but elusive goal. Unbeknown to us, however, myocyte regeneration may accomplish just that. Continuous cell renewal in the adult myocardium was thought to be impossible, but multipotent cardiac stem cells may be able to renew the myocardium and, under certain circumstances, can be coaxed to repair the broken heart after infarction. (Heart Views 2003;4(1): pp? © 2003 Gulf Heart Association


 

Introduction

   Cardiac myocytes are thought to be terminally differentiated cells and have been often compared to neurons for their inability to regenerate and replace damaged myocardium. Even though evidence now exists for adult neurogenesis and neural stem cells (1), the concept of myocyte regeneration has not been embraced by the medical community and remains highly disputed (2). Hypertrophy has been assumed to be the only form of myocyte growth in the heart, and over the years information has been gathered on the many signalling pathways implicated in myocyte hypertrophy (3). Conversely, the mechanisms of myocyte regeneration have been mostly neglected.
   Difficulties in interpreting cellular labelling experiments, owing to the complexity of identifying myocytes that are duplicating DNA by conventional light microscopy, have limited analysis of myocyte regeneration. But recent results in humans and animals have provided evidence that myocyte replication does occur under physiological and pathological conditions of the heart (4-6). The use of Ki67 and 5-bromodeoxyuridine (BrdU) as markers of cell proliferation, together with the use of contractile protein antibodies for recognizing myocytes, has allowed us to identify multiplying myocytes by high-resolution confocal microscopy (4-6). In spite of these observations, however, the skepticism about myocyte division remains so strong that the upregulation of cyclins, cyclindependent kinases (CDKs) and telomerase activity in the heart have been viewed as biochemical events of cellular hypertrophy (7,8) rather than indices of cell proliferation.


 

Myocyte replication and the infarcted heart

   Evidence suggesting that some myocytes divide comes from several studies in animal models that show that myocytes express early and late growth-related genes immediately after infarction. Quantities of cyclin E, A, and B are increased and their associated kinase activities are elevated significantly (4). In addition, high levels of DNA replication, karyokinesis and cytokinesis have been identified (4-6). These aspects of ventricular remodelling have been shown to occur after infarction, and in other models of heart failure in which mitotic indices have been measured in myocardial sections and dissociated myocytes (5,9).
   The concept of multiple myocyte divisions in the mammalian heart has been strengthened by the recognition of telomeric shortening and the decrease of telomerase activity (10,11) in the decompensated ageing rat heart. However, acute heart failure in dogs is characterized by cell regeneration with preservation of telomeric length, owing to a marked increase in the activity of telomerase (12).

Fig. 1. Myocyte division. a-c, Stages of mitosis in human myocytes. Metaphase chromosomes (a), karyokinesis (b) and cytokinesis (c) are shown by the green fluorescence of propidium iodide (PI). Red fluorescence reflects cardiac myosin antibody staining of myocyte cytoplasm. Arrows indicate mitosis. Scale bars, 10 µ m. d, Changes in myocyte growth in acute and chronic infarcts. MI, myocardial infarct.

   Studies of the postinfarcted human heart shortly after coronary occlusion and late during the terminal stages of the ischemic myopathy have characterized the effects of time on the extent of myocyte replication (5,6). Notably, myocytes in mitosis are present in control hearts, which suggests that myocyte regeneration contributes to the homeostasis of the nondiseased heart. Cell growth is markedly enhanced acutely after infarction and more in the border zone than in the remote tissue. The number of dividing myocytes (Fig. 1a-c) is 3 - 4-fold higher at 1 week after infarction (6) than in end-stage cardiac failure, years after the primary event (6). Because myocytes in the infarcted area die in a few hours and ischemic damage destroys the vascular and nonvascular components of the interstitium, formation of new myocardium in the infarcted region through myocyte growth alone would seem to be impossible. Cell proliferation occurs exclusively in the border zone and in distant tissue where the blood supply is largely maintained (13).
   Quantitative results consistent with myocyte proliferation have been frequently based on the assumption that myocytes are mononucleated cells (2,4). Thus, an increase in the total number of myocyte nuclei would correspond to an identical increase in the number of cells in the ventricle. But different proportions of mononucleated and binucleated myocytes may be present, and this possibility complicates the distinction between karyokinesis and cytokinesis. Karyokinesis without cytokinesis results in an increase in the number of nuclei per cell, but the actual number of cells remains constant. Conversely, cytokinesis produces an increase in the number of cells, whereas the number of nuclei per cell does not change. Importantly, the human heart is composed of nearly 80% mononucleated and 20% binucleated ventricular myocytes, and this ratio is not altered by sex, ageing, cardiac hypertrophy or ischemic cardiomyopathy (14).
   Together, these data support the notion that a subpopulation of adult myocytes re-enter the cell cycle and proliferate. Myocyte regeneration and cellular hypertrophy constitute the growth reserve of the heart and can expand significantly the functioning myocardium after infarction (Fig.1d). Thus, the concept that myocardial infarction represents a demonstration of the terminally differentiated state of myocytes should be reconsidered.


 

Origin of replicating myocytes

   The identification of cycling cells in the myocardium as myocytes is based on their expression of myocyte-specific markers. To facilitate their recognition, interstitial cells are not labelled and appear as scattered nuclei (Fig. 1a-c). Components of the contractile apparatus have been the most commonly used indicators of the origin of these dividing cells (4-6). Myocytes in mitosis may be rich in organized myofibrils that occupy a predominant portion of the cytoplasm (6). These characteristics indicate that there are mature myocytes in the adult human heart that have the ability to re-enter the cell cycle and divide. However, the extent of the replicative potential of myocytes is unknown.

Fig. 2. Primitive cells and myocyte ageing. Cells are positive (green) for c-kit (a), MDR1 (b) and Sca-1 (c) in rat ventricular myocardium. Cardiac myosin is stained red, PI is blue. The c-kit-labelled cell is in mitosis (a). Scale bars, 10 µ m.

   There are two possible origins of cycling myocytes. First, these cells might be part of a small pool of replicating myoblasts that divide asymmetrically and continuously generate new myocytes for normal homeostasis or in response to pathological stimuli. But these cells do not grow in culture, which, together with the absence of evidence for clonal expansion in vivo in BrdU-labelling experiments in rats (16) makes the existence of such a population unlikely. Second, the dividing myocytes might be amplifying cells derived from stem cells that expand and produce a differentiated progeny under proper stimulation. This progeny could represent the cycling myocytes that after a few cell divisions withdraw from the cell cycle and become terminally differentiated, reaching growth arrest (6).
   An important issue is whether myocyte precursors derive from resident cardiac stem cells (CSCs) or from circulating stem cells that have homed to the heart. Undifferentiated cells expressing stem-cell-related antigens have been identified in the adult myocardium. Our unpublished data show that primitive cells can be detected by three surface markers (Fig.2): c-kit, which is the receptor (16,17) for stem cell factor; MDR1, which is a P-glycoprotein capable of extruding dyes, toxic substances and drugs (18,19); and Sca-1, which is involved in cell signalling and cell adhesion (20). None of these markers is specific for stem cells (Fig.3), as each one is found in hematopoietic stem cells and other cell types 21-24). Whether these cells are CSCs remains to be ascertained. But Lin-c-kitPOS bone marrow cells can reconstitute mouse myocardium in vivo (25,26).
   An issue of biological and clinical relevance is whether newly formed myocytes are derived from CSCs that accumulated in the heart early in development or are the progeny of hematopoietic stem cells that, later in life, home to the myocardium from the systemic circulation. In favor of the first possibility is the fact that c-kitPOS cells migrate during fetal growth and form colonies in several organs, including the heart (27,28). Chemotaxis of hematopoietic stem cells is modulated by stem cell factor, which promotes their migration to specific sites (29), suggesting that stem cells may have been stored in the heart as remnants from the cardiac primordia. These primitive cells may have undergone symmetric and asymmetric division (30), expanding the pool size of undifferentiated cells in the maturing heart.

Fig. 3. Structure, distribution and function of surface markers of stem cells.

   The recent studies by us and our co-workers (25,26) and others (31-33) showing that bone marrow cells (BMCs) can regenerate myocardium after infarction establish that blood-borne cells can acquire properties of CSCs and differentiate.
   Further support for the role of adult cycling myocytes in ventricular remodelling has come from the recognition that 20% of these cells in dogs express telomerase (12). Many telomerase-competent myocytes are cycling, as indicated by the presence of Ki67 protein in their nuclei; however, the number of divisions may be limited. Telomeres constitute the physical ends of chromosomes, and telomerase can keep the length of telomeres intact after each cell cycle (34,35). This prevents premature senescence and sustains cell multiplication (36). Progenitor cells and rapidly replicating, amplifying cells have high levels of telomerase activity (37). Telomerase has been recognized not only in myocytes but also in neurons from organs with low turnover rates (12,38).

Myocyte ageing, volume and growth

   It remains a general belief that the number of myocytes in the heart is defined at birth and these cells persist throughout life. There are men and women 100 years old and older, and this fact would imply that all of their myocytes have lived 100 years or more – in other words, the age of individuals and the age of their myocytes should coincide. But myocytes do not live indefinitely; they have a limited lifespan in humans and rodents (39,40). Cell loss and myocyte proliferation are part and parcel of normal homeostasis, and an increase in these parameters is typical of cardiac ageing (41).
   The old heart is characterized by a reduction in cell number and hypertrophy of the remaining myocytes (41), and this phenotype has been used to argue against the formation of new myocytes. But without cell regeneration the rates of cell death (4,41) would imply that all myocytes would die during the first few months of a rodent’s lifespan (40). For example, the left ventricle of a young rat contains 13 X 106 myocytes, and at any point in time 200 and 93,000 myocytes are dying by apoptosis and necrosis, respectively. Because apoptosis is completed in nearly 4 h and necrosis in roughly 24 h, 94,200 myocytes are lost in one day. Thus, 2.83 X 106 cells would die in 1 month, and the total 13 X 106 ventricular myocytes would disappear in 5 months.
   Throughout life, a mixture of young and old cells is present in the rat myocardium. Although most myocytes seem to be terminally differentiated, there is a fraction of younger myocytes (15–20%) that retains the capacity to replicate (10,11). The proportion of these two populations changes with age, and there is no point in life at which all myocytes are comparable in terms of age, size, shape and molecular properties. Whether a myocyte responds to pathological loads by hypertrophy or replication is influenced by cell volume, which in turn reflects its age. Large cells are old, do not react to growth stimuli, and are more prone to activate cell death. Small cells are younger, can re-enter the cell cycle or hypertrophy, and are less susceptible to death.
   There is a good correlation between myocyte age and expression of the CDK inhibitor p16INK4a (p16), which has proved to be a marker of cellular ageing (42). p16 is detectable in 10% of myocytes at birth, and in more than 80% of myocytes in senescent rats (10). Telomeric length follows a similar pattern (longer in younger, smaller cells and shorter in older, larger ones), reflecting a decrease of telomerase with age (10,11). The age-dependent increase in myocyte death, coupled with a reduction in coronary vasculature, may explain why myocardial infarction is associated with increased mortality in the elderly (43).


 

Cellular therapy for myocardial infarction

   New therapies of myocardial infarction include the implantation of skeletal myoblasts and bone-marrow-derived cardiomyocytes (44,46); however, these strategies have failed to reconstitute healthy myocardium (46). The growth potential of adult BMCs offers a promising new tool. These cells home to the infarcted region by local injection (25), by mobilization by cytokines (26) or by spontaneous translocation after injury (32).
   Homed BMCs proliferate and differentiate into myocytes, smooth muscle cells and endothelial cells, resulting in the partial regeneration of the destroyed myocardium (25,26). In addition, the growth response mediated by BMCs interferes with ventricular scarring and decompensation (25,26). Small myocytes and vascular structures develop in the infarct and mostly replace the dead zone (Fig.4). For the first time, BMCs have been shown to generate myocardium in vivo, thus reducing infarct size (25,26). Contraction reappears in the area of injury, diastolic stress is decreased, ventricular haemodynamics is improved and mortality is reduced (26). The new myocytes are more reminiscent of fetal than adult cells, however, and the chronic evolution of the reconstituted heart remains to be determined.

Fig. 4. Myocardial repair. a. Band of regenerating myocardium after infarction (arrowheads). b,c. Newly formed small myocytes (b) and coronary arterioles (c) in the developing band. Cardiac myosin (a,b) and smooth muscle actin (c) and are stained red; PI is yellow-green. Scale bars: 100 µm (a); 10 µm (b,c).

   But should BMCs be considered to be the cells of choice for cardiac repair or could CSCs be used to replace necrotic myocardium? CSCs might be more effective than BMCs in rebuilding dead tissue. BMCs have to re-programme themselves to give rise to progeny differentiating into cardiac lineages; by contrast, activation and migration of CSCs to the site of injury would avoid this intermediate phase. Moreover, CSCs may be faster than BMCs in reaching functional competence and structural characteristics of mature myocytes and vessels. This approach would require the recognition of growth factors that affect CSCs in a discrete and restrictive manner, and do not stimulate BMCs or stem cells in other organs. The identification of cardiac cells with surface markers of stem cells (Fig. 2) is consistent with this possibility. The high degree of myocyte regeneration in the non-infarcted myocardium in humans (6) supports this hypothesis. Recruitment and expansion of CSCs would promote a pool of young replicating myocytes and neovascularization.

Future directions

   Here we have outlined several new findings that provide an alternative view of myocardial biology that might one day lead to a reconsideration of the long-term goals of the therapy of myocardial infarction and chronic heart failure. This outlook advances the possibility that it might be feasible to develop strategies for the actual regeneration of the infarcted ventricle.
   In terms of the controversial proposal that the normal heart undergoes a continuous turnover of myocytes that increases under pathological conditions, we need to understand better the origin and behaviour of cycling myocytes to maximize myocardial growth and cardiac repair. New data suggest that CSCs exist and are the source of tissue renewal. The identification, localization and purification of CSCs, and the characterization of CSC biology, are essential issues that must be resolved. This may lead to the discovery of mediators of CSC migration, proliferation and differentiation that, in turn, might result in the mending of the “broken heart.”

Supported by NIH Grant

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Reprinted by permission. Nature 2002;415:240- 243. © Copyright 2002 Macmillan Publishers Ltd.


   

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