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Innate Regeneration in the Aging Heart: Healing From Within

  • Piero Anversa
    Correspondence
    Correspondence: Address to Piero Anversa, MD, Department of Anesthesia, Department of Medicine, and Division of Cardiovascular Medicine, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis St, Boston, MA 02115.
    Affiliations
    Department of Anesthesia, Department of Medicine, and Division of Cardiovascular Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA
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  • Annarosa Leri
    Affiliations
    Department of Anesthesia, Department of Medicine, and Division of Cardiovascular Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA
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      Abstract

      The concept of the heart as a terminally differentiated organ incapable of replacing damaged myocytes has been at the center of cardiovascular research and therapeutic development for the past 50 years. The progressive decline in myocyte number as a function of age and the formation of scarred tissue after myocardial infarction have been interpreted as irrefutable proofs of the postmitotic characteristic of the heart. However, emerging evidence supports a more dynamic view of the heart in which cell death and renewal are vital components of the remodeling process that governs cardiac homeostasis, aging, and disease. The identification of dividing myocytes in the adult and senescent heart raises the important question concerning the origin of these newly formed cells. In vitro and in vivo findings strongly suggest that replicating myocytes derive from lineage determination of resident primitive cells, supporting the notion that cardiomyogenesis is controlled by activation and differentiation of a stem cell compartment. It is the current view that the myocardium is an organ permissive of tissue regeneration mediated by exogenous and endogenous progenitor cells.

      Abbreviations and Acronyms:

      CSC (cardiac stem cell), EF (ejection fraction), hCSC (human cardiac stem cell)
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      Learning Objectives: Educational objectives. On completion of this article, you should be able to (1) establish whether the human heart is a postmitotic organ or a self-renewing organ regulated by a resident cardiac stem cell compartment, (2) determine whether the aging myopathy is a stem cell disease, and (3) evaluate whether autologous cardiac stem cells can be used therapeutically in patients with chronic heart failure.
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      A fundamental issue pertaining to the ability of the heart to sustain cardiac diseases of ischemic and nonischemic origin is whether myocardial regeneration occurs in the adult organ or whether this growth adaptation is restricted to prenatal life, severely limiting the response of the myocardium to pathologic stresses.
      • Anversa P.
      • Leri A.
      • Kajstura J.
      Cardiac regeneration.
      The concept of the heart as a terminally differentiated organ incapable of replacing damaged myocytes has been at the center of cardiovascular research for the past 50 years.
      • Murry C.E.
      • Field L.J.
      • Menasché P.
      Cell-based cardiac repair: reflections at the 10-year point.
      • Laflamme M.A.
      • Murry C.E.
      Regenerating the heart.
      • Rubart M.
      • Field L.J.
      Cardiac regeneration: repopulating the heart.
      • Hansson E.M.
      • Lindsay M.E.
      • Chien K.R.
      Regeneration next: toward heart stem cell therapeutics.
      • Yi B.A.
      • Wernet O.
      • Chien K.R.
      Pregenerative medicine: developmental paradigms in the biology of cardiovascular regeneration.
      The accepted view has been that the postnatal, adult, and aging heart reacts to an increase in workload by hypertrophy of existing myocytes only. Hypertrophied cardiomyocytes express the senescence-associated proteins p16INK4a and p53
      • Urbanek K.
      • Quaini F.
      • Tasca G.
      • et al.
      Intense myocyte formation from cardiac stem cells in human cardiac hypertrophy.
      • Urbanek K.
      • Torella D.
      • Sheikh F.
      • et al.
      Myocardial regeneration by activation of multipotent cardiac stem cells in ischemic heart failure.
      • Kajstura J.
      • Gurusamy N.
      • Ogórek B.
      • et al.
      Myocyte turnover in the aging human heart.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Role of cardiac stem cells in cardiac pathophysiology: a paradigm shift in human myocardial biology.
      • Anversa P.
      • Kajstura J.
      • Rota M.
      • Leri A.
      Regenerating new heart with stem cells.
      and are prone to undergo apoptosis and necrosis, possibly because of the high intracellular content of reactive oxygen species. Cell death is restricted to p16INK4a-positive myocytes, but the process is inefficient, resulting in the progressive accumulation of poorly functional myocytes
      • Kajstura J.
      • Gurusamy N.
      • Ogórek B.
      • et al.
      Myocyte turnover in the aging human heart.
      ; they are characterized by changes in the expression of contractile protein isoforms and by profound alterations in intracellular calcium cycling and electrical properties.
      • Rota M.
      • Hosoda T.
      • De Angelis A.
      • et al.
      The young mouse heart is composed of myocytes heterogeneous in age and function.
      • Bernhard D.
      • Laufer G.
      The aging cardiomyocyte: a mini-review.
      • Shih H.
      • Lee B.
      • Lee R.J.
      • Boyle A.J.
      The aging heart and post-infarction left ventricular remodeling.
      These defects, together with myocyte loss and the development of foci of myocardial scarring, inevitably contribute in the long term to the onset of ventricular dysfunction and its progression to overt failure.

      Regenerative Capacity of Adult Organs

      The ability of stem cells to continuously replenish the compartment of undifferentiated and lineage-committed cells is a typical property of higher organisms with mitotic soma. In these cases, tissues are capable of renewal and repair, and the extent of regeneration positively correlates with animal lifespan.
      • Maier B.
      • Gluba W.
      • Bernier B.
      • et al.
      Modulation of mammalian life span by the short isoform of p53.
      • Hosoda T.
      • Rota M.
      • Kajstura J.
      • Leri A.
      • Anversa P.
      Role of stem cells in cardiovascular biology.
      The reduced longevity of lower organisms, including Caenorhabditis elegans and Drosophila, is linked to the lack of regenerative potential of their postmitotic tissues in adulthood.
      • Maier B.
      • Gluba W.
      • Bernier B.
      • et al.
      Modulation of mammalian life span by the short isoform of p53.
      Dying cells cannot be replaced, leading to a rapid decline in organ function. Conversely, cell turnover by proliferation and commitment of resident progenitor cells is active in mammals, and old injured cells lost as a result of normal wear and tear may be restored by new, better-functioning cells. However, regeneration is hampered in the presence of damage, which creates a barrier to restitutio ad integrum and promotes a repair process leading to the formation of a scar.
      • Anversa P.
      • Leri A.
      • Kajstura J.
      Cardiac regeneration.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Cardiac stem cells and mechanisms of myocardial regeneration.
      The scar does not possess the biochemical, physical, and functional properties of the intact tissue, negatively affecting the remodeling of the diseased heart, a critical determinant of cardiac performance.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Cardiac stem cells and mechanisms of myocardial regeneration.
      • Pfeffer M.A.
      • Braunwald E.
      Ventricular remodeling after myocardial infarction: experimental observations and clinical implications.
      Despite the presence of resident adult stem cells, the repair mechanism triggered by ischemic injury results in collagen accumulation and scarring. This phenomenon is not restricted to the heart but occurs in all the organs, whether their parenchymal cells are highly proliferating, slowly cycling, or terminally differentiated. This intrinsic limitation was interpreted as the inability of the adult myocardium to generate de novo cardiomyocytes.
      • Hosoda T.
      • Rota M.
      • Kajstura J.
      • Leri A.
      • Anversa P.
      Role of stem cells in cardiovascular biology.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Cardiac stem cells and mechanisms of myocardial regeneration.
      However, the outcome of infarction is essentially identical throughout the organism. Occlusion of a major conductive artery or a large branch results in loss of tissue in the skin, kidney, intestine, brain, liver, and reproductive organs in a manner identical to the heart.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Cardiac stem cells and mechanisms of myocardial regeneration.
      • Mori N.
      • Horie Y.
      • Nimura Y.
      • Wolf R.
      • Granger D.N.
      Hepatic microvascular responses to ischemia-reperfusion in low-density lipoprotein receptor knockout mice.
      • Mori A.
      • Hashino S.
      • Kobayashi S.
      • et al.
      Avascular necrosis in the femoral head secondary to bone marrow infarction in a patient with graft-versus-host disease after unrelated bone marrow transplantation.
      • Narayan S.
      • Ezughah F.
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      • Kennedy C.T.
      Idiopathic hypereosinophilic syndrome associated with cutaneous infarction and deep venous thrombosis.
      • Francque S.
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      • Asselah T.
      • et al.
      Multifactorial aetiology of hepatic infarction: a case report with literature review.
      • Leong F.T.
      • Freeman L.J.
      Acute renal infarction.
      This common outcome is conditioned by 2 crucial factors: (1) stem cells in the infarct die as do all other cells deprived of oxygen supply and (2) resident stem cells activate a local regenerative response but do not migrate from the viable tissue to the damaged area to replace the necrotic tissue (Figure 1).
      Figure thumbnail gr1
      Figure 1Heart failure and myocardial regeneration. A, Area of regenerating myocardium in the infarct. Proliferating, small, developing myocytes are positive for the cell-cycle marker MCM5 (white). Myocytes are labeled by cardiac myosin (red) and nuclei by DAPI (blue). Two small myocytes in mitosis are shown in the insets (magnification 300X). B, The cluster of cells included in a rectangle in the middle of an acute infarct is shown at higher magnification in C and D. These cells express c-kit (green, arrows) and at times cardiac myosin (red; D), reflecting undifferentiated cardiac stem cells and early committed progenitors, respectively. MI = myocardial infarction. Scale bars: A and B, 100 μm; C and D, 10 μm.
      The main factors that dictate the evolution of a lesion into scarring rather than regeneration have not been identified with certainty. Some insights come from embryonic development. Repair in embryos is rapid, efficient, and scar free.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Cardiac stem cells and mechanisms of myocardial regeneration.
      Skin wounds in the early mammalian embryo heal with restitutio ad integrum, whereas wounds in late gestational fetuses and adult mammals result in scarring. This difference may be related to the intrinsic characteristics of embryonic fibroblasts or to external stimuli. Fibroblasts in embryos are less prone to synthesize and release collagen, attenuating the amount of fibrosis after injury.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Cardiac stem cells and mechanisms of myocardial regeneration.
      • Mackool R.J.
      • Gittes G.K.
      • Longaker M.T.
      Scarless healing: the fetal wound.
      The presence of hyaluronic acid and fibronectin in the amniotic fluid may also interfere with scar formation during wound healing. Moreover, the inflammatory response is attenuated; a low number of poorly differentiated inflammatory cells accumulate in the region of damage, and the growth factors present at the site of healing are different from those in the adult organism.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Cardiac stem cells and mechanisms of myocardial regeneration.
      • Ferguson M.W.
      • O'Kane S.
      Scar-free healing: from embryonic mechanisms to adult therapeutic intervention.
      • Larson B.J.
      • Longaker M.T.
      • Lorenz H.P.
      Scarless fetal wound healing: a basic science review.
      • Leung A.
      • Crombleholme T.M.
      • Keswani S.G.
      Fetal wound healing: implications for minimal scar formation.
      Scar-free regeneration of damaged organs is well known in amphibians and zebra fish.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Cardiac stem cells and mechanisms of myocardial regeneration.
      • Nye H.L.
      • Cameron J.A.
      • Chernoff E.A.
      • Stocum D.L.
      Regeneration of the urodele limb: a review.
      Epimorphic regeneration corresponds to the complete restoration of organs after the development of body plans and cellular differentiation.
      • Hosoda T.
      • Rota M.
      • Kajstura J.
      • Leri A.
      • Anversa P.
      Role of stem cells in cardiovascular biology.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Cardiac stem cells and mechanisms of myocardial regeneration.
      • Hata S.
      • Namae M.
      • Nishina H.
      Liver development and regeneration: from laboratory study to clinical therapy.
      However, this occurs only in invertebrates and lower vertebrates, including urodeles and fish. Conversely, mammals retain limited capacity for spontaneous regeneration in the adult liver and infant fingertips,
      • Hosoda T.
      • Rota M.
      • Kajstura J.
      • Leri A.
      • Anversa P.
      Role of stem cells in cardiovascular biology.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Cardiac stem cells and mechanisms of myocardial regeneration.
      • Hata S.
      • Namae M.
      • Nishina H.
      Liver development and regeneration: from laboratory study to clinical therapy.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Mechanisms of myocardial regeneration.
      suggesting that during evolution animals gradually lose their ability to reconstitute damaged organs. Characterization of the processes that drive regeneration in lower vertebrates may provide some information for the implementation of regenerative strategies in humans. Regeneration of the adult heart occurs in newts and zebra fish and has been claimed to involve the reentry of cardiomyocytes into the cell cycle
      • Kikuchi K.
      • Holdway J.E.
      • Werdich A.A.
      • et al.
      Primary contribution to zebrafish heart regeneration by gata4(+) cardiomyocytes.
      • Kikuchi K.
      • Poss K.D.
      Cardiac regenerative capacity and mechanisms.
      or dedifferentiation.
      • Anversa P.
      • Kajstura J.
      • Rota M.
      • Leri A.
      Regenerating new heart with stem cells.
      Dedifferentiation refers to a regression of a mature cell in its own lineage; this process is typically coupled with attainment of the proliferative capacity. Dedifferentiation is considered the natural regenerative response to cardiac injury in zebra fish, newts, and planaria. After disassembly of the sarcomeric contractile apparatus, which impedes cytokinesis, differentiated cardiomyocytes replicate to reconstitute up to 20% of the zebra fish ventricle after resection.
      • Anversa P.
      • Kajstura J.
      • Rota M.
      • Leri A.
      Regenerating new heart with stem cells.
      By cre/lox cell lineage analysis, the regenerating myocardium has been shown to derive primarily from a subpopulation of GATA4-positive cardiomyocytes rather than from nonmyocyte sources, such as stem cells. The poorly organized sarcomeric structure of replicating GATA4-positive myocytes has led to the hypothesis that dedifferentiation constitutes the primary regenerative step.
      • Kikuchi K.
      • Poss K.D.
      Cardiac regenerative capacity and mechanisms.
      However, the failure in the induction of the early myocyte commitment genes Nkx2.5 and Hand2 in the replicating myocytes indicate that the cells of origin underwent limited dedifferentiation or, most likely, that replicating GATA4-positive cardiomyocytes correspond to cells at the late stage of commitment of stem cell differentiation. In the mouse heart, Nkx2.5 expression precedes GATA4 in transit-amplifying cardiomyocytes originated by lineage specification of cardiac stem cells (CSCs) (Figure 2).
      • Boni A.
      • Urbanek K.
      • Nascimbene A.
      • et al.
      Notch1 regulates the fate of cardiac progenitor cells.
      Figure thumbnail gr2a
      Figure 2Notch1 up-regulates Nkx2.5 but not GATA4 during early commitment of cardiac stem cells (CSCs). A, Hes1 transcript was measured in CSCs at 2, 5, and 8 days under control conditions (Ctrl) and in the presence of Jagged1 (Jag1) and Jag1 and γ-secretase inhibitor (Jag1+γ). Hes1 quantity is shown as fold changes vs the corresponding Ctrl. B, After Jag1 stimulation, CSCs are c-kit negative and display nuclear localization of the active fragment of Notch1 (Notch1 intracellular domain [N1ICD], yellow). One CSC continues to express c-kit (green) together with the extracellular domain of the Notch1 receptor (red, arrow). C and D, Jag1-stimulated CSCs express N1ICD (green) together with Nkx2.5 (red). The area included in the square is shown at higher magnification in the inset (magnification: 1000X). E and F, After γ-secretase inhibition, Jag1-stimulated CSCs are negative for N1ICD and Nkx2.5. G, Percentage of CSCs positive for N1ICD and Nkx2.5 at 2, 5, and 8 days. H and I, The expression of GATA4 (yellow) is shown in CSCs stimulated by Jag1 in the (H) absence and (I) presence of γ-secretase inhibitor. J, Percentage of CSCs positive for GATA4 at 5 days. K, Nkx2.5 and GATA4 transcripts were analyzed in CSCs at 5 days. mRNA = messenger RNA. P<.05 vs Ctrl. ∗∗P<.05 vs Jag1. ∗∗∗Statistically different vs both Ctrl and Jag1. Data are shown as mean ± SD.
      Figure thumbnail gr2b
      Figure 2Notch1 up-regulates Nkx2.5 but not GATA4 during early commitment of cardiac stem cells (CSCs). A, Hes1 transcript was measured in CSCs at 2, 5, and 8 days under control conditions (Ctrl) and in the presence of Jagged1 (Jag1) and Jag1 and γ-secretase inhibitor (Jag1+γ). Hes1 quantity is shown as fold changes vs the corresponding Ctrl. B, After Jag1 stimulation, CSCs are c-kit negative and display nuclear localization of the active fragment of Notch1 (Notch1 intracellular domain [N1ICD], yellow). One CSC continues to express c-kit (green) together with the extracellular domain of the Notch1 receptor (red, arrow). C and D, Jag1-stimulated CSCs express N1ICD (green) together with Nkx2.5 (red). The area included in the square is shown at higher magnification in the inset (magnification: 1000X). E and F, After γ-secretase inhibition, Jag1-stimulated CSCs are negative for N1ICD and Nkx2.5. G, Percentage of CSCs positive for N1ICD and Nkx2.5 at 2, 5, and 8 days. H and I, The expression of GATA4 (yellow) is shown in CSCs stimulated by Jag1 in the (H) absence and (I) presence of γ-secretase inhibitor. J, Percentage of CSCs positive for GATA4 at 5 days. K, Nkx2.5 and GATA4 transcripts were analyzed in CSCs at 5 days. mRNA = messenger RNA. P<.05 vs Ctrl. ∗∗P<.05 vs Jag1. ∗∗∗Statistically different vs both Ctrl and Jag1. Data are shown as mean ± SD.
      A comparable regenerative response has been observed after surgical resection of the apex of the left ventricle in the neonatal mouse heart.
      • Porrello E.R.
      • Mahmoud A.I.
      • Simpson E.
      • et al.
      Transient regenerative potential of the neonatal mouse heart.
      Again, cardiomyocyte proliferation was considered as the cellular adaptation supporting the repair process. Genetic fate mapping strategies have been used to distinguish the contribution of preexisting myocytes and stem/progenitor cells to regeneration of the zebra fish and mouse heart. This approach, however, has several intrinsic limitations. The specificity of the promoter and its transactivated protein for the population of cells to be studied is an essential prerequisite of lineage tracing. Cardiac stem cells engineered with fluorescent constructs placed under the control of promoters encoding myocyte-specific transcription factors and sarcomeric proteins show transgene expression.
      • Bailey B.
      • Izarra A.
      • Alvarez R.
      • et al.
      Cardiac stem cell genetic engineering using the alphaMHC promoter.
      Although GATA4 and α-myosin heavy chain promoters are generally considered to be active primarily in cardiomyocytes, the presence of the reporter gene in c-kit–positive CSCs reflects a very early stage of cardiogenic lineage commitment of these mother cells, which, after the loss of the stem cell antigen, give rise to a large compartment of transit-amplifying myocytes (Figure 3). Thus, the lack of specificity severely hampers the validity of the findings obtained with fate mapping strategies when promoters encode alleged myocyte-restricted proteins.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Role of cardiac stem cells in cardiac pathophysiology: a paradigm shift in human myocardial biology.
      • Anversa P.
      • Kajstura J.
      • Rota M.
      • Leri A.
      Regenerating new heart with stem cells.
      Figure thumbnail gr3
      Figure 3Transit-amplifying myocytes. Small developing myocytes in the infarct are positive for (A) telomerase (magenta) and MCM5 (white) and for (B) MEF2C (yellow) and connexin 43 (green, arrowheads). TERT = telomerase reverse transcriptase. Scale bars, 10 mm.
      The essential foundation of meticulously performed lineage tracing protocols consists of expression of the fluorescent label in the pool of cells hierarchically located upstream of the progeny to be tracked; the use of promoters regulating contractile proteins introduces a serious bias into the method. The availability of cre/lox mice carrying fluorescent tags under the control of stem cell antigens will clarify this controversial issue. Data collected in mice with spontaneous inactive mutations of c-kit and mice carrying a deletion of Sca-1 strongly suggest that stem cells contribute significantly to cardiomyogenesis during homeostasis and regulate the magnitude of myocyte regeneration after injury.
      • Hosoda T.
      • Yasuzawa-Amano S.
      • Amano K.
      • et al.
      Defects in the c-kit-SCF system have profound effects on myocardial gene expression.
      • Bastos Carvalho A.
      • Carrillo-Infante C.
      • D'Amario D.
      • et al.
      Mutations of c-kit receptor are coupled with impaired growth and enhanced death of cardiac progenitor cells.
      • Bailey B.
      • Fransioli J.
      • Gude N.A.
      • et al.
      Sca-1 knockout impairs myocardial and cardiac progenitor cell function.
      Thus, current observations in zebra fish and neonatal, adult, and old mice do not exclude that CSCs participate in myocardial growth.
      Evidence of dedifferentiation, ie, reentry of terminally differentiated cardiomyocytes into the cell cycle, is based exclusively on cellular morphology. Cells characterized by an intermediate phenotype between forming and mature myocytes have arbitrarily been considered the product of dedifferentiation.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Role of cardiac stem cells in cardiac pathophysiology: a paradigm shift in human myocardial biology.
      • Anversa P.
      • Kajstura J.
      • Rota M.
      • Leri A.
      Regenerating new heart with stem cells.
      • Kubin T.
      • Pöling J.
      • Kostin S.
      • et al.
      Oncostatin M is a major mediator of cardiomyocyte dedifferentiation and remodeling.
      However, the lack of structural markers typical of a given cell type is not by itself indicative of the developmental stage of the cell
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Role of cardiac stem cells in cardiac pathophysiology: a paradigm shift in human myocardial biology.
      ; it is impossible to define in vivo whether the cell of interest is undergoing differentiation or whether it is in the process of reverting to an earlier immature state. As discussed previously herein, the possibility of dedifferentiation was raised by observations conducted in vertebrates with exceptional regenerative abilities. This conclusion, however, has recently been challenged. During salamander limb reconstitution, cells from muscle, bone, cartilage, nerve sheath, and connective tissues are believed to dedifferentiate into a pool of proliferating cells known as the regeneration blastema.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Cardiac regeneration and aging.
      • Leri A.
      • Anversa P.
      • Kajstura J.
      Stem cells and heart disease.
      • Sugimoto K.
      • Gordon S.P.
      • Meyerowitz E.M.
      Regeneration in plants and animals: dedifferentiation, transdifferentiation, or just differentiation?.
      But the possibility that preexisting unipotent or multipotent stem cells constitute the pool of proliferating blastema cells
      • Kragl M.
      • Roensch K.
      • Nüsslein I.
      • et al.
      Muscle and connective tissue progenitor populations show distinct Twist1 and Twist3 expression profiles during axolotl limb regeneration.
      questions the notion of dedifferentiation and subsequent redifferentiation of a common pool of cells responsible for the repair of the amputated limb.
      • Kragl M.
      • Roensch K.
      • Nüsslein I.
      • et al.
      Muscle and connective tissue progenitor populations show distinct Twist1 and Twist3 expression profiles during axolotl limb regeneration.
      Similarly, the hypothesis has been raised that clonally dominant cardiomyocytes with properties similar to those of stem cells direct heart morphogenesis in zebra fish from the early stages of development to adulthood.
      • Gupta V.
      • Poss K.D.
      Clonally dominant cardiomyocytes direct heart morphogenesis.

      Myocardial Regeneration: The Controversy

      A critical issue of cardiac pathobiology concerns whether myocardial regeneration occurs in the adult organ or whether this growth adaptation is restricted to prenatal life and ceases shortly after birth.
      • Anversa P.
      • Leri A.
      • Kajstura J.
      Cardiac regeneration.
      • Murry C.E.
      • Field L.J.
      • Menasché P.
      Cell-based cardiac repair: reflections at the 10-year point.
      • Laflamme M.A.
      • Murry C.E.
      Regenerating the heart.
      • Rubart M.
      • Field L.J.
      Cardiac regeneration: repopulating the heart.
      • Hansson E.M.
      • Lindsay M.E.
      • Chien K.R.
      Regeneration next: toward heart stem cell therapeutics.
      • Yi B.A.
      • Wernet O.
      • Chien K.R.
      Pregenerative medicine: developmental paradigms in the biology of cardiovascular regeneration.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Role of cardiac stem cells in cardiac pathophysiology: a paradigm shift in human myocardial biology.
      • Anversa P.
      • Kajstura J.
      • Rota M.
      • Leri A.
      Regenerating new heart with stem cells.
      The concept that the heart is a postmitotic, terminally differentiated organ unable to replace its myocyte compartment has been at the center of cardiovascular research for several decades.
      • Anversa P.
      • Leri A.
      • Kajstura J.
      Cardiac regeneration.
      • Murry C.E.
      • Field L.J.
      • Menasché P.
      Cell-based cardiac repair: reflections at the 10-year point.
      • Laflamme M.A.
      • Murry C.E.
      Regenerating the heart.
      • Rubart M.
      • Field L.J.
      Cardiac regeneration: repopulating the heart.
      • Hansson E.M.
      • Lindsay M.E.
      • Chien K.R.
      Regeneration next: toward heart stem cell therapeutics.
      • Yi B.A.
      • Wernet O.
      • Chien K.R.
      Pregenerative medicine: developmental paradigms in the biology of cardiovascular regeneration.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Role of cardiac stem cells in cardiac pathophysiology: a paradigm shift in human myocardial biology.
      • Anversa P.
      • Kajstura J.
      • Rota M.
      • Leri A.
      Regenerating new heart with stem cells.
      • MacLellan W.R.
      • Schneider M.D.
      Genetic dissection of cardiac growth control pathways.
      The accepted view is that the myocardium reacts to an increased workload by hypertrophy of the existing myocytes, and when this cellular response is exhausted, ventricular dysfunction supervenes. Based on this assumption, molecular cardiology has focused in the past 30 years on identification of the multiple signaling pathways regulating the activation and depression of genes implicated in the hypertrophic growth of cardiomyocytes during postnatal maturation or after abnormal pathologic states or chronological aging.
      • Anversa P.
      • Leri A.
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      Cardiac regeneration.
      • Leri A.
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      Role of cardiac stem cells in cardiac pathophysiology: a paradigm shift in human myocardial biology.
      • Anversa P.
      • Kajstura J.
      • Rota M.
      • Leri A.
      Regenerating new heart with stem cells.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Cardiac stem cells and mechanisms of myocardial regeneration.
      • MacLellan W.R.
      • Schneider M.D.
      Genetic dissection of cardiac growth control pathways.
      • Anversa P.
      • Kajstura J.
      Ventricular myocytes are not terminally differentiated in the adult mammalian heart.
      • Soonpaa M.H.
      • Field L.J.
      Survey of studies examining mammalian cardiomyocyte DNA synthesis.
      • Chien K.R.
      • Olson E.N.
      Converging pathways and principles in heart development and disease: [email protected]
      • Oh H.
      • Schneider M.D.
      The emerging role of telomerase in cardiac muscle cell growth and survival.
      • Chien K.R.
      • Karsenty G.
      Longevity and lineages: toward the integrative biology of degenerative diseases in heart, muscle, and bone.
      The possibility that the heart can renew its parenchymal cells has been largely dismissed, and, even today, myocardial repair is viewed with suspicion and trepidation.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Cardiac regeneration and aging.
      • Leri A.
      • Anversa P.
      • Kajstura J.
      Stem cells and heart disease.
      The engrained paradigm that promotes a rather uninteresting biological perspective of the developing, old, and diseased heart has been shaken by a variety of studies indicating that myocyte regeneration occurs in humans and animals after infarction,
      • Urbanek K.
      • Torella D.
      • Sheikh F.
      • et al.
      Myocardial regeneration by activation of multipotent cardiac stem cells in ischemic heart failure.
      • Beltrami A.P.
      • Urbanek K.
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      • et al.
      Evidence that human cardiac myocytes divide after myocardial infarction.
      after prolonged pressure overload,
      • Urbanek K.
      • Quaini F.
      • Tasca G.
      • et al.
      Intense myocyte formation from cardiac stem cells in human cardiac hypertrophy.
      and in the decompensated senescent heart.
      • Olivetti G.
      • Ricci R.
      • Anversa P.
      Hyperplasia of myocyte nuclei in long-term cardiac hypertrophy in rats.
      • Chimenti C.
      • Kajstura J.
      • Torella D.
      • et al.
      Senescence and death of primitive cells and myocytes lead to premature cardiac aging and heart failure.
      Although some of these studies were published almost 25 years ago
      • Anversa P.
      • Palackal T.
      • Sonnenblick E.H.
      • Olivetti G.
      • Meggs L.G.
      • Capasso J.M.
      Myocyte cell loss and myocyte cellular hyperplasia in the hypertrophied aging rat heart.
      and systematically continued to appear in the literature (for reviews, see references
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Role of cardiac stem cells in cardiac pathophysiology: a paradigm shift in human myocardial biology.
      ,
      • Anversa P.
      • Kajstura J.
      • Rota M.
      • Leri A.
      Regenerating new heart with stem cells.
      ,
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Cardiac regeneration and aging.
      , and
      • Leri A.
      • Anversa P.
      • Kajstura J.
      Stem cells and heart disease.
      ), the traditional establishment tends to reject this alternative notion of cardiac biology, defending a territory that was considered unwavering and immovable.
      The recognition that myocyte death by apoptosis and necrosis occurs continuously in the adult heart of humans and animals
      • Kajstura J.
      • Gurusamy N.
      • Ogórek B.
      • et al.
      Myocyte turnover in the aging human heart.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Cardiac stem cells and mechanisms of myocardial regeneration.
      • Torella D.
      • Rota M.
      • Nurzynska D.
      • et al.
      Cardiac stem cell and myocyte aging, heart failure, and insulin-like growth factor-1 overexpression.
      • Gonzalez A.
      • Rota M.
      • Nurzynska D.
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      Activation of cardiac progenitor cells reverses the failing heart senescent phenotype and prolongs lifespan.
      emphasizes the necessity for an equivalent degree of myocyte formation to preserve cardiac mass and function. The discovery of resident CSCs and their ability to differentiate into cardiomyocytes, vascular endothelial cells, and smooth muscle cells has provoked a rather negative response in part of the scientific community; CSCs were either ignored or claimed to lack biological significance.
      • Hansson E.M.
      • Lindsay M.E.
      • Chien K.R.
      Regeneration next: toward heart stem cell therapeutics.
      • Murry C.E.
      • Lee R.T.
      Development biology: turnover after the fallout.
      • Parmacek M.S.
      • Epstein J.A.
      Cardiomyocyte renewal.
      Similarly, the documentation that hematopoietic stem cells transdifferentiate, generating cardiomyocytes and coronary vessels after infarction,
      • Anversa P.
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      Cardiac regeneration.
      • Orlic D.
      • Kajstura J.
      • Chimenti S.
      • et al.
      Bone marrow cells regenerate infarcted myocardium.
      • Rota M.
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      • Hosoda T.
      • et al.
      Bone marrow cells adopt the cardiomyogenic fate in vivo.
      was immediately rejected, prompting a series of negative studies that challenged the validity of the early observations.
      • Murry C.E.
      • Soonpaa M.H.
      • Reinecke H.
      • et al.
      Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts.
      • Balsam L.B.
      • Wagers A.J.
      • Christensen J.L.
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      • Robbins R.C.
      Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium.
      Some comments about the history of the heart as a postmitotic organ may be relevant for understanding the shift in paradigm required for the implementation of the novel field of regenerative cardiology.

      The Heart as a Postmitotic Organ

      Numerous studies of the human heart from 1850 to 1911 held the view that myocardial hypertrophy was the consequence of hyperplasia and hypertrophy of existing myocytes.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Role of cardiac stem cells in cardiac pathophysiology: a paradigm shift in human myocardial biology.
      • Anversa P.
      • Kajstura J.
      Ventricular myocytes are not terminally differentiated in the adult mammalian heart.

      Forester A. Handbuch der Speciellen Patholgischen Anatomie. Leipzig, Germany: 1852.

      • Zielonko G.
      Pathologisch anatomie und experiementelle studie uber hypertrophie des herzens.
      • Wideroe S.
      Histologische studien uber die muskulatur des herzens.
      However, subsequent reports from 1921 to 1925 questioned the ability of myocytes to proliferate, suggesting that the increase in cardiac muscle mass in the pathologic heart was the result of pure cellular hypertrophy.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Role of cardiac stem cells in cardiac pathophysiology: a paradigm shift in human myocardial biology.
      • Anversa P.
      • Kajstura J.
      Ventricular myocytes are not terminally differentiated in the adult mammalian heart.
      • Aschoff L.
      Pathologische Anatomie.

      Kaufmann E. Specielle Pathologische Anatomie. Berlin/Leipzig, Germany: 1922.

      • Karsner H.T.
      • Saphir O.
      • Todd T.W.
      The state of the cardiac muscle in hypertrophy and atrophy.
      The concept that myocytes cannot divide originated from difficulty in identifying mitotic figures in these cells in the absence of quantitative evaluations of myocyte size and number.
      • Karsner H.T.
      • Saphir O.
      • Todd T.W.
      The state of the cardiac muscle in hypertrophy and atrophy.
      This conviction gained support from autoradiographic analysis of thymidine incorporation in the hearts of animals during postnatal growth
      • Petersen R.O.
      • Baserga R.
      Nucleic acid and protein synthesis in cardiac muscle of growing and adult mice.
      and after pressure overload.
      • Morkin E.
      • Ashford T.P.
      Myocardial DNA synthesis in experimental cardiac hypertrophy.
      • Grove D.
      • Nair K.G.
      • Zak R.
      Biochemical correlates of cardiac hypertrophy, 3: changes in DNA content; the relative contributions of polyploidy and mitotic activity.
      • Grove D.
      • Zak R.
      • Nair K.G.
      • Aschenbrenner V.
      Biochemical correlates of cardiac hypertrophy, IV: observations on the cellular organization of growth during myocardial hypertrophy in the rat.
      DNA synthesis in myocyte nuclei was not detected, prompting the conclusion that the heart survives and exerts its function until the death of the organism with the same cells that are present at birth.
      • Zak R.
      Development and proliferative capacity of cardiac muscle cells.
      Accordingly, ventricular myocytes in humans are terminally differentiated cells, and their lifespan corresponds to the lifespan of the organism. The number of myocytes attains an adult value a few months after birth,
      • Anversa P.
      • Olivetti G.
      Cellular basis of physiologic and pathologic myocardial growth.
      and the same myocytes are believed to contract 70 times per minute throughout life. Because a certain fraction of the population reaches 100 years of age or more, an inevitable consequence of this paradigm is that cardiac myocytes are immortal, functionally and structurally.
      • Anversa P.
      • Leri A.
      • Kajstura J.
      Cardiac regeneration.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Role of cardiac stem cells in cardiac pathophysiology: a paradigm shift in human myocardial biology.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      Cardiac stem cells and mechanisms of myocardial regeneration.
      • Anversa P.
      • Kajstura J.
      Ventricular myocytes are not terminally differentiated in the adult mammalian heart.
      In view of this conviction, we discuss myocardial aging and the potential mechanisms involved in acquisition of the cardiac senescent phenotype. In addition, we address the differences in the rate of myocyte turnover claimed by various laboratories and the potential role that resident human CSCs have in organ homeostasis and regeneration as a function of age. This information is critical to define whether the aging myopathy is a stem cell disease, and strategies can be developed to modify the progression of ventricular dysfunction in the old heart.
      • Anversa P.
      • Rota M.
      • Urbanek K.
      • et al.
      Myocardial aging: a stem cell problem.

      Myocardial Aging in Humans

      The aging myopathy typically manifests itself with diastolic dysfunction and preserved ejection fraction (EF).
      • Aurigemma G.P.
      Diastolic heart failure: a common and lethal condition by any name.
      More than 50% of patients with heart failure have normal or near-normal EF, and the incidence and prevalence of this condition increases with age.
      • Aurigemma G.P.
      Diastolic heart failure: a common and lethal condition by any name.
      • Kitzman D.W.
      • Gardin J.M.
      • Gottdiener J.S.
      • et al.
      Cardiovascular Health Study Research Group
      Importance of heart failure with preserved systolic function in patients > or = 65 years of age.
      • Aurigemma G.P.
      • Gaasch W.H.
      Clinical practice: diastolic heart failure.
      • Bhatia R.S.
      • Tu J.V.
      • Lee D.S.
      • et al.
      Outcome of heart failure with preserved ejection fraction in a population-based study.
      • Owan T.E.
      • Hodge D.O.
      • Herges R.M.
      • Jacobsen S.J.
      • Roger V.L.
      • Redfield M.M.
      Trends in prevalence and outcome of heart failure with preserved ejection fraction.
      Although the claim is commonly made that age-associated physiologic changes predispose older adults to development of heart failure with a normal EF, the etiology of diastolic heart failure is unknown. The difficulty in defining myocardial aging and the mechanisms involved further complicates recognition of the cellular processes underlying impaired diastolic relaxation. Morbidity and mortality for chronic heart failure continue to increase and parallel the extension in median lifespan of the population,
      • Sanderson W.C.
      • Scherbov S.
      Average remaining lifetimes can increase as human populations age.
      pointing to aging as the major risk factor of the human disease. At present, there are 5.1 million patients with chronic heart failure in the United States alone, with an incidence of 670,000 new cases per year,
      • Roger V.L.
      • Go A.S.
      • Lloyd-Jones D.M.
      • et al.
      Heart disease and stroke statistics—2011 update: a report from the American Heart Association.
      and most individuals with chronic heart failure are 65 years or older.
      Currently, we have little understanding of the etiology of myocardial aging. Rarely, studies in animals and humans have considered aging as an independent process and time as the major cause of the manifestations of the aging myopathy. Aging has been interpreted as a variable, which cooperates with a variety of diseases, to define the old heart.
      • Lakatta E.G.
      • Levy D.
      Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises, part II: the aging heart in health: links to heart disease.
      • Lakatta E.G.
      Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises, part III: cellular and molecular clues to heart and arterial aging.
      • Hadley E.C.
      • Lakatta E.G.
      • Morrison-Bogorad M.
      • Warner H.R.
      • Hodes R.J.
      The future of aging therapies.
      Only occasional reports have characterized cardiac aging in humans independent of concomitant pathologic states.
      • Kajstura J.
      • Gurusamy N.
      • Ogórek B.
      • et al.
      Myocyte turnover in the aging human heart.
      • Chimenti C.
      • Kajstura J.
      • Torella D.
      • et al.
      Senescence and death of primitive cells and myocytes lead to premature cardiac aging and heart failure.
      • Kajstura J.
      • Rota M.
      • Cappetta D.
      • et al.
      Cardiomyogenesis in the aging and failing human heart.
      Myocardial aging differs in women and men, emphasizing a sex difference in the adaptive response of the myocardium to physiologic aging alone or together with cardiovascular diseases. Diastolic heart failure predominates in old women,
      • Roger V.L.
      • Go A.S.
      • Lloyd-Jones D.M.
      • et al.
      Heart disease and stroke statistics—2011 update: a report from the American Heart Association.
      and, although it is frequently observed in men, systolic and diastolic function is impaired more commonly in old men. The senescent male heart has an attenuated ability to sustain increases in pressure load, possibly mediated by the decrease in the number of myocytes early in life, which increases with age.
      • Urbanek K.
      • Torella D.
      • Sheikh F.
      • et al.
      Myocardial regeneration by activation of multipotent cardiac stem cells in ischemic heart failure.
      • Olivetti G.
      • Melissari M.
      • Capasso J.M.
      • Anversa P.
      Cardiomyopathy of the aging human heart: myocyte loss and reactive cellular hypertrophy.
      • Olivetti G.
      • Giordano G.
      • Corradi D.
      • et al.
      Gender differences and aging: effects on the human heart.
      Conversely, myocyte number remains constant in women up to nearly 90 years of age,
      • Olivetti G.
      • Giordano G.
      • Corradi D.
      • et al.
      Gender differences and aging: effects on the human heart.
      and this difference may explain the higher incidence of systolic-diastolic heart failure in men.
      • Gottdiener J.S.
      • Arnold A.M.
      • Aurigemma G.P.
      • et al.
      Predictors of congestive heart failure in the elderly: the Cardiovascular Health Study.

      Myocardial Aging and CSCs

      The concept of myocardial aging is complex, and this difficulty is dictated by the identification of parameters that define the consequences of time alone on the heart independent of cardiac and noncardiac pathologic abnormalities. Cardiac aging has been confounded by the notion that the heart is a postmitotic organ characterized by a predetermined number of myocytes, which is established at birth and preserved until the death of the organ and organism.
      • Murry C.E.
      • Field L.J.
      • Menasché P.
      Cell-based cardiac repair: reflections at the 10-year point.
      • Laflamme M.A.
      • Murry C.E.
      Regenerating the heart.
      • Rubart M.
      • Field L.J.
      Cardiac regeneration: repopulating the heart.
      • Hansson E.M.
      • Lindsay M.E.
      • Chien K.R.
      Regeneration next: toward heart stem cell therapeutics.
      According to this old paradigm, the generation of myocytes is restricted to the fetal neonatal heart, and organ hypertrophy in the adult occurs only by myocyte enlargement. Based on this premise, cellular, organ, and organism age coincide; at any given time, the heart is composed of a homogeneous population of myocytes of identical age. Because of this static view, aging has been construed as a time-dependent process that interacts with ischemic injury, hypertension, diabetes, and other disorders, which together define the clinical phenotype.
      • Anversa P.
      • Rota M.
      • Urbanek K.
      • et al.
      Myocardial aging: a stem cell problem.
      • Roger V.L.
      • Go A.S.
      • Lloyd-Jones D.M.
      • et al.
      Heart disease and stroke statistics—2011 update: a report from the American Heart Association.
      • Lakatta E.G.
      • Levy D.
      Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises, part II: the aging heart in health: links to heart disease.
      • Lakatta E.G.
      Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises, part III: cellular and molecular clues to heart and arterial aging.
      The discovery that stem cells live in the heart and differentiate into the various cardiac cell lineages has dramatically changed our understanding of myocardial biology.
      • Beltrami A.P.
      • Barlucchi L.
      • Torella D.
      • et al.
      Adult cardiac stem cells are multipotent and support myocardial regeneration.
      • Linke A.
      • Müller P.
      • Nurzynska D.
      • et al.
      Stem cells in the dog heart are self-renewing, clonogenic, and multipotent and regenerate infarcted myocardium, improving cardiac function.
      • Bearzi C.
      • Rota M.
      • Hosoda T.
      • et al.
      Human cardiac stem cells.
      • Bearzi C.
      • Leri A.
      • Lo Monaco F.
      • et al.
      Identification of a coronary vascular progenitor cell in the human heart.
      • Hosoda T.
      • D'Amario D.
      • Cabral-Da-Silva M.C.
      • et al.
      Clonality of mouse and human cardiomyogenesis in vivo.
      • Oh H.
      • Bradfute S.B.
      • Gallardo T.D.
      • et al.
      Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction.
      • Pfister O.
      • Mouquet F.
      • Jain M.
      • et al.
      CD31- but not CD31+ cardiac side population cells exhibit functional cardiomyogenic differentiation.
      • Tomita Y.
      • Matsumura K.
      • Wakamatsu Y.
      • et al.
      Cardiac neural crest cells contribute to the dormant multipotent stem cell in the mammalian heart.
      • Oyama T.
      • Nagai T.
      • Wada H.
      • et al.
      Cardiac side population cells have a potential to migrate and differentiate into cardiomyocytes in vitro and in vivo.
      • Smith R.R.
      • Barile L.
      • Cho H.C.
      • et al.
      Regenerative potential of cardiosphere-derived cells expanded from percutaneous endomyocardial biopsy specimens.
      The CSCs are multipotent cells capable of generating cardiomyocytes and coronary vessels, providing the missing link between the identification of small dividing myocytes and the uncertainty concerning the origin of these repopulating cells (see references
      • Leri A.
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      Role of cardiac stem cells in cardiac pathophysiology: a paradigm shift in human myocardial biology.
      ,
      • Leri A.
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      Cardiac stem cells and mechanisms of myocardial regeneration.
      ,
      • Anversa P.
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      Life and death of cardiac stem cells: a paradigm shift in cardiac biology.
      , and
      • Anversa P.
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      Concise review: stem cells, myocardial regeneration, and methodological artifacts.
      ). Most importantly, this information has imposed a reconsideration of our view of cardiac homeostasis and myocardial aging. A new paradigm has emerged: the heart is a self-renewing organ characterized by resident CSCs stored in niches (Figure 4). The niches control the physiologic turnover of cardiac cells and the growth, migration, and commitment of CSCs that leave the niches, replacing dying cells in the myocardium.
      • Boni A.
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      Notch1 regulates the fate of cardiac progenitor cells.
      • Urbanek K.
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      Stem cell niches in the adult mouse heart.
      • Hosoda T.
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      Human cardiac stem cell differentiation is regulated by a mircrine mechanism.
      • Goichberg P.
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      The ephrin A1-EphA2 system promotes cardiac stem cell migration after infarction.
      This information has imposed a reconsideration of myocardial aging.
      Figure thumbnail gr4
      Figure 4Cardiac niches in the human heart. A-C, Cluster of c-kit–positive cardiac stem cells (CSCs) (green). Arrows in A define the areas shown at higher magnification in B and C (magnification 1600X). Gap junctions (connexin 43 [Cx43], white; arrowheads) and adherens junctions (N-cadherin [N-cadh], magenta; arrowheads) are illustrated. Connexin 43 and N-cadh are present between CSCs and myocytes (α-sarcomeric actin [SA], red) and fibroblasts (procollagen, light blue; asterisks). Fibronectin, yellow. qdot = quantum dots.
      In the past, myocardial aging has been attributed to genetic modifications, accumulation of oxidative DNA damage, mutations in mitochondrial DNA, and telomeric dysfunction, triggering growth arrest, cellular senescence, and death.
      • Aubert G.
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      Telomeres and aging.
      • Haigis M.C.
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      The aging stress response.
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      Aging and disease as modifiers of efficacy of cell therapy.
      • Blanpain C.
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      DNA-damage response in tissue-specific and cancer stem cells.
      • Collado M.
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      Cellular senescence in cancer and aging.
      Although these determinants of organ aging are all valid, the cellular target in the heart may have changed. Aging effects on myocytes, smooth muscle cells, endothelial cells, and fibroblasts may be only an epiphenomenon dictated by CSC aging. Organ aging may reflect a process in which temporal alterations in CSC behavior determine the phenotypic properties of the myocardium. Myocardial aging may be regulated by time-dependent changes in the biology of CSCs. Growth defects at the level of the controlling cell, ie, the CSC, may perturb the structural integrity and function of the old heart. Old myocytes nested in proximity to CSCs may secrete a variety of inflammatory molecules and factors favoring cellular senescence and apoptosis, altering the physiologic behavior of stem cell niches in the myocardium.
      • Liu L.
      • Rando T.A.
      Manifestations and mechanisms of stem cell aging.
      A typical example is provided by the enhanced synthesis and release of angiotensin II from hypertrophied cardiomyocytes,
      • Leri A.
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      • et al.
      Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell.
      • Leri A.
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      • et al.
      Up-regulation of AT(1) and AT(2) receptors in postinfarcted hypertrophied myocytes and stretch-mediated apoptotic cell death.
      • Leri A.
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      • Wang X.
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      Overexpression of insulin-like growth factor-1 attenuates the myocyte renin-angiotensin system in transgenic mice.
      • Leri A.
      • Liu Y.
      • Claudio P.P.
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      Insulin-like growth factor-1 induces Mdm2 and down-regulates p53, attenuating the myocyte renin-angiotensin system and stretch-mediated apoptosis.
      resulting in the activation of angiotensin II type 1 receptors present in CSCs.
      • Gonzalez A.
      • Rota M.
      • Nurzynska D.
      • et al.
      Activation of cardiac progenitor cells reverses the failing heart senescent phenotype and prolongs lifespan.
      • D'Amario D.
      • Cabral-Da-Silva M.C.
      • Zheng H.
      • et al.
      Insulin-like growth factor-1 receptor identifies a pool of human cardiac stem cells with superior therapeutic potential for myocardial regeneration.
      This process is commonly coupled with initiation of the apoptotic pathway. The effects of time on CSCs are critical for understanding the mechanisms conditioning CSC senescence, myocyte and vascular aging, and, ultimately, cardiac aging and organism lifespan.
      The telomere-telomerase axis is a determinant of cellular aging. Human CSCs (hCSCs) have telomeres of approximately 10 kilobase pairs (kbp), and with each cell division there is a loss of approximately 130 base pairs of telomeric DNA.
      • Bearzi C.
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      Human cardiac stem cells.
      Telomere length reflects the past replicative history and cumulative oxidative DNA damage occurring during the life cycle of the cell.
      • Aubert G.
      • Lansdorp P.M.
      Telomeres and aging.
      • Dimmeler S.
      • Leri A.
      Aging and disease as modifiers of efficacy of cell therapy.
      • Collado M.
      • Blasco M.A.
      • Serrano M.
      Cellular senescence in cancer and aging.
      Telomerase activity delays but cannot prevent telomere erosion, which is mediated by down-regulation of telomerase, formation of reactive oxygen species, and loss of telomere-related proteins.
      • Haigis M.C.
      • Yankner B.A.
      The aging stress response.
      When telomere length reaches 1.5 to 2.0 kbp, replicative senescence and irreversible growth arrest occur.
      • Vaziri H.
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      • Allsopp R.C.
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      Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age.
      The presence of telomere-induced dysfunction foci in hCSCs is coupled with expression of the senescence-associated proteins p16INK4a and p53
      • Cesselli D.
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      • et al.
      Effects of age and heart failure on human cardiac stem cell function.
      and, eventually, initiation of the apoptotic pathway (Figure 5).
      Figure thumbnail gr5
      Figure 5Cardiac stem cell (CSC) senescence. A-F, Two examples each of c-kit–positive CSCs (green) expressing (A and B) p16INK4a (yellow) and (C and D) p53 (magenta) are shown. E and F, Similarly, telomeres in c-kit–positive CSCs are illustrated in 2 examples by small white fluorescence dots. Arrows indicate c-kit–positive CSCs. Scale bars, 10 μm.
      Growth activation of hCSCs with a wide range of telomere lengths leads to a progeny with distinct phenotypes. Chronological age results in telomere attrition of hCSCs, and these cells form myocytes that rapidly express p16INK4a.
      • Kajstura J.
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      • et al.
      Myocyte turnover in the aging human heart.
      Daughter cells inherit the shortened telomeres of the maternal cells, and after a few rounds of division and terminal differentiation, they typically show an old cell phenotype.
      • Kajstura J.
      • Gurusamy N.
      • Ogórek B.
      • et al.
      Myocyte turnover in the aging human heart.
      • Gonzalez A.
      • Rota M.
      • Nurzynska D.
      • et al.
      Activation of cardiac progenitor cells reverses the failing heart senescent phenotype and prolongs lifespan.
      Telomere length in hCSCs and human myocytes shows a consistent pattern; if hCSCs exhibit long telomeres, the myocytes formed possess comparable telomere length. Conversely, hCSCs with short telomeres generate a myocyte progeny with severe telomere attrition. This phenomenon affects the aging female and male human heart; however, the proportion of hCSCs/myocytes with short telomeres is higher in men than in women, and the fraction of hCSCs/myocytes with long telomeres is larger in women than in men.
      • Kajstura J.
      • Gurusamy N.
      • Ogórek B.
      • et al.
      Myocyte turnover in the aging human heart.
      Telomere length in hCSCs defines cellular aging independent of chronological age and organism lifespan. Oncogenic stress
      • Rodier F.
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      may be viewed as an additional stimulus that accelerates senescence in amplifying myocytes. Oncogenic proliferative signals are coupled with several growth inhibitory responses generally seen with the induction of cellular aging and activation of the endogenous cell death pathway.
      • Urbanek K.
      • Rota M.
      • Cascapera S.
      • et al.
      Cardiac stem cells possess growth factor-receptor systems that after activation regenerate the infarcted myocardium, improving ventricular function and long-term survival.
      Insulinlike growth factor 1 delays cardiac aging and rescues the infarcted myocardium experimentally,
      • Torella D.
      • Rota M.
      • Nurzynska D.
      • et al.
      Cardiac stem cell and myocyte aging, heart failure, and insulin-like growth factor-1 overexpression.
      • Gonzalez A.
      • Rota M.
      • Nurzynska D.
      • et al.
      Activation of cardiac progenitor cells reverses the failing heart senescent phenotype and prolongs lifespan.
      • Urbanek K.
      • Rota M.
      • Cascapera S.
      • et al.
      Cardiac stem cells possess growth factor-receptor systems that after activation regenerate the infarcted myocardium, improving ventricular function and long-term survival.
      • Li Q.
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      although it fails to initiate the division of terminally differentiated postmitotic myocytes. This parenchymal cell compartment may react with abortive mitosis
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      or the formation of anaphase bridges.

      Function of hCSCs

      Human beings up to 104 years of age possess a significant number of hematopoietic stem cells with long telomeres and remarkable growth reserve that, however, are in a quiescent state.
      • Kajstura J.
      • Gurusamy N.
      • Ogórek B.
      • et al.
      Myocyte turnover in the aging human heart.
      Conversely, most activated hCSCs in the senescent myocardium have short telomeres that are inherited by the specialized progeny. In the young healthy heart, the asymmetrical kinetics of stem cell growth efficiently preserves organ homeostasis. The structural and functional decline of the old and diseased heart may be coupled with defects in the hierarchical growth of the organ, suggesting that quantitative and qualitative alterations occur in resident hCSCs or in the pool of transit-amplifying cardiomyocytes. Both possibilities have been documented in stem cell–regulated organs.
      The number of epithelial stem cells in the small intestine is gradually reduced with age,
      • Martin K.
      • Kirkwood T.B.
      • Potten C.S.
      Age changes in stem cells of murine small intestinal crypts.
      and the function of hematopoietic stem cells changes with time.
      • Rossi D.J.
      • Bryder D.
      • Zahn J.M.
      • et al.
      Cell intrinsic alterations underlie hematopoietic stem cell aging.
      Whether aging of the skin is conditioned by a decreased frequency of epidermal stem cells or by alterations in the kinetics of the transit-amplifying cell pool remains controversial.
      • Martin K.
      • Kirkwood T.B.
      • Potten C.S.
      Age changes in stem cells of murine small intestinal crypts.
      • Blanpain C.
      • Fuchs E.
      Epidermal homeostasis: a balancing act of stem cells in the skin.
      Similarly, the environmental and cell-autonomous mechanisms that maintain “young” hCSCs in a state of long-term quiescence remain to be identified. However, the existence of a pool of hCSCs with intact telomeres, 8 to 12 kbp, in senescent female and male hearts and in explanted hearts
      • Kajstura J.
      • Gurusamy N.
      • Ogórek B.
      • et al.
      Myocyte turnover in the aging human heart.
      • Cesselli D.
      • Beltrami A.P.
      • D'Aurizio F.
      • et al.
      Effects of age and heart failure on human cardiac stem cell function.
      is of great clinical relevance. This category of hCSCs with high growth reserve is expected to generate a young myocyte progeny in the failing and senescent heart. Because each division of hCSCs results in the loss of approximately 130 base pairs of telomeric DNA,
      • Bearzi C.
      • Leri A.
      • Lo Monaco F.
      • et al.
      Identification of a coronary vascular progenitor cell in the human heart.
      an extremely large number of cardiomyocytes can be formed by these cells before critical telomeric shortening and growth arrest occur. From a clinical perspective, the recognition that a subset of telomerase-competent hCSCs with long telomeres persists at all ages and with chronic heart failure has raised the possibility that autologous cell-based therapy may be feasible in patients with severe ventricular dysfunction. Recently, a method has been developed to isolate this compartment of functionally competent hCSCs from endomyocardial biopsies of patients undergoing cardiac transplant or left ventricular assist device implantation.
      • D'Amario D.
      • Fiorini C.
      • Campbell P.M.
      • et al.
      Functionally competent cardiac stem cells can be isolated from endomyocardial biopsies of patients with advanced cardiomyopathies.
      After in vitro amplification, a clinically relevant number of hCSCs with high myogenic and vasculogenic potential was obtained. Expanded hCSCs possess a significant growth reserve as documented by the short population doubling time, high telomerase activity, and relatively long telomeres.
      The phase 1 trial SCIPIO (Stem Cell Infusion in Patients with Ischemic cardiOmyopathy) involves the delivery of autologous c-kit–positive lineage-negative hCSCs for the treatment of severe chronic heart failure of ischemic origin.
      • Bolli R.
      • Chugh A.R.
      • D'Amario D.
      • et al.
      Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised phase 1 trial.
      • Chugh A.R.
      • Beache G.M.
      • Loughran J.H.
      • et al.
      Administration of cardiac stem cells in patients with ischemic cardiomyopathy: the SCIPIO trial: surgical aspects and interim analysis of myocardial function and viability by magnetic resonance.
      Patients with EF lower than 40% at 4 months after coronary artery bypass grafting were enrolled in the treatment and control groups. Treated patients received a single intracoronary infusion of 1 million autologous hCSCs. The primary end point was short-term treatment safety, and the secondary end point was efficacy. No hCSC-related adverse effects were reported. In 14 CSC-treated patients who were analyzed, EF increased from 30% to 38% at 4 months after infusion. In contrast, 7 control patients, during the corresponding interval, did not show any change in this functional parameter. The beneficial effects of CSCs were even more pronounced at 1 year. In 7 treated patients, infarct size decreased 24% and 30% at 4 and 12 months, respectively.
      • Bolli R.
      • Chugh A.R.
      • D'Amario D.
      • et al.
      Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised phase 1 trial.
      • Chugh A.R.
      • Beache G.M.
      • Loughran J.H.
      • et al.
      Administration of cardiac stem cells in patients with ischemic cardiomyopathy: the SCIPIO trial: surgical aspects and interim analysis of myocardial function and viability by magnetic resonance.
      These initial results are encouraging and warrant further studies.

      Conclusion

      The human heart is a highly dynamic organ regulated by a pool of resident hCSCs that modulate cardiac homeostasis and condition organ aging. Hopefully, recent findings will resolve the debate that has divided the scientific community into strong opponents and passionate supporters of the regenerative potential of the human heart. A common ground can now be found to translate this different perspective of cardiac biology into the development of novel strategies for the management of the human disease.

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