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The paper by Roberto Bolli and colleagues (Nov 26, p 1847) on cardiac progenitor cell treatment for ischaemic heart failure is a milestone in the history of cardiac cell therapy. However, we noticed some points that possibly interfere with interpretation of the results. First, the trial is still ongoing, so why did Bolli and colleagues publish partial results at this time? Why not wait until the trial was completed? Second, 13 analysed patients were from the non-randomised stage of the trial—only ten had undergone random allocation. Moreover, the ratio of treated to control patients (24:32) was reversed (7:3) by the analysis stage, mainly because of patients’ withdrawal. Why did so many control patients lose interest in the study? We are cautious because of our own experience. When one of us (CS) started a cell therapy programme using CD133+ cells delivered during coronary artery bypass graft (CABG) surgery, a striking improvement in heart function was noted in the initial pilot trial. A partly randomised, open-label trial later showed a clear benefi t of the concomitant cell therapy over CABG surgery alone. Finally, we did a strictly double-blind, fully randomised, placebo-controlled trial (CARDIO133, NCT00462774) and recently analysed the results. Unfortunately, none of the previous positive eff ects were confi rmed. Essentially, we followed the wrong track for nearly 10 years, because we did not strictly adhere to the guidelines on how to do a randomised, controlled clinical trial. We hope this will not be the fate of the SCIPIO concept.

Transplantation of culture-expanded adult stem/progenitor cells often results in poor cellular engraftment, survival, and migration into sites of tissue injury. Mesenchymal cells including fibroblasts and stromal cells secrete factors that protect injured tissues, promote tissue repair, and support many types of stem/progenitor cells in culture. We hypothesized that secreted factors in conditioned medium (CdM) from adult bone marrow-derived multipotent stromal cells (MSCs) could be used to prime adult cardiac stem/progenitor cells (CSCs/CPCs) and improve graft success after myocardial infarction (MI). Incubation of adult rat CPCs in CdM from human MSCs isolated by plastic adherence or by magnetic sorting against CD271 (a.k.a., p75 low-affinity nerve growth factor receptor; p75MSCs) induced phosphorylation of STAT3 and Akt in CPCs, supporting their proliferation under normoxic conditions and survival under hypoxic conditions (1% oxygen). Priming CSCs with 30× p75MSC CdM for 30 minutes prior to transplantation into subepicardial tissue 1 day after MI markedly increased engraftment compared with vehicle priming. Screening CdM with neutralizing/blocking antibodies identified connective tissue growth factor (CTGF) and Insulin as key factors in p75MSC CdM that protected CPCs. Human CTGF peptide (CTGF-D4) and Insulin synergistically promoted CPC survival during hypoxia in culture. Similar to CdM priming, priming of CSCs with CTGF-D4 and Insulin for 30 minutes prior to transplantation promoted robust engraftment, survival, and migration of CSC derivatives at 1 week and 1 month after MI. Our results indicate that short-term priming of human CSCs with CTGF-D4 and Insulin may improve graft success and cardiac regeneration in patients with MI. Stem Cells 2014;32:674–683

Background— Little is known about the function of inositol 1,4,5-trisphosphate receptors (IP3Rs) in the adult heart experimentally. Moreover, whether these Ca 2+ release channels are present and play a critical role in human cardiomyocytes remains to be defined. IP3Rs may be activated after Gαq-protein–coupled receptor stimulation, affecting Ca 2+ cycling, enhancing myocyte performance, and potentially favoring an increase in the incidence of arrhythmias. Methods and Results— IP3R function was determined in human left ventricular myocytes, and this analysis was integrated with assays in mouse myocytes to identify the mechanisms by which IP3Rs influence the electric and mechanical properties of the myocardium. We report that IP3Rs are expressed and operative in human left ventricular myocytes. After Gαq-protein–coupled receptor activation, Ca 2+ mobilized from the sarcoplasmic reticulum via IP3Rs contributes to the decrease in resting membrane potential, prolongation of the action potential, and occurrence of early afterdepolarizations. Ca 2+ transient amplitude and cell shortening are enhanced, and extrasystolic and dysregulated Ca 2+ elevations and contractions become apparent. These alterations in the electromechanical behavior of human cardiomyocytes are coupled with increased isometric twitch of the myocardium and arrhythmic events, suggesting that Gαq-protein–coupled receptor activation provides inotropic reserve, which is hampered by electric instability and contractile abnormalities. Additionally, our findings support the notion that increases in Ca 2+ load by IP3Rs promote Ca 2+ extrusion by forward-mode Na + /Ca 2+ exchange, an important mechanism of arrhythmic events. Conclusions— The Gαq-protein/coupled receptor/IP3R axis modulates the electromechanical properties of the human myocardium and its propensity to develop arrhythmias.

The lack of myocardial repair after myocardial infarction and the heart failure that eventually ensues was thought of as proof that myocardial cell regeneration and myocardial repair mechanisms do not exist. Recently, growing experimental and clinical evidence has proven this concept wrong. Cardiac stem cells and endogenous myocardial repair mechanisms do exist; however, they do not produce significant myocardial repair. Similarly, the preliminary results of stem cell therapy for myocardial repair have shown early promise but modest results. Preclinical studies are the key to understanding stem cell senescence and lack of cellular contact and vasculature in the infarcted region. Additional laboratory studies are sure to unlock the therapeutic mechanisms that will be required for significant myocardial repair.

Rationale:Various types of viable stem cells have been reported to result in modest improvement in cardiac function after acute myocardial infarction. The mechanisms for improvement from different stem cell populations remain unknown. Objective:To determine whether irradiated (nonviable) embryonic stem cells (iESCs) improve postischemic cardiac function without adverse consequences. Methods and Results:After coronary artery ligation-induced cardiac infarction, either conditioned media or male murine or male human iESCs were injected into the penumbra of ischemic myocardial tissue of female mice or female rhesus macaque monkeys, respectively. Murine and human iESCs, despite irradiation doses that prevented proliferation and induced cell death, significantly improved cardiac function and decreased infarct size compared with untreated or media-treated controls. Fluorescent in situ hybridization of the Y chromosome revealed disappearance of iESCs within the myocardium, whereas 5-bromo-2′-deoxyuridine assays r...

A Subpopulation of Adult Skeletal Muscle Stem Cells Retains All Template DNA Strands After Cell Division Rocheteau et al Cell . 2012;148:112–125. Adult skeletal muscle stem cells are a heterogeneous cell population characterized by a small subset of undifferentiated cells that express at high levels the paired/homeodomain gene Pax7 . This category of satellite cells divides predominantly by asymmetric chromatid segregation, generating a daughter cell that carries the mother DNA and retains stem cell property, and a daughter cell that inherits the newly synthesized DNA and acquires the myocyte lineage.1 Recently, the established modalities of stem cell self-renewal, that is, generation of a daughter stem cell and a daughter-committed cell, have been questioned, and an old theory that may have important implications in stem cell function and senescence has been reconsidered. The possibility that nonrandom chromatid segregation regulates stem cell division has gained interest, promoting intense discussion in the field.2–6 The immortal DNA strand hypothesis advanced by John Cairns in 19752 suggests that stem cell division is characterized by asymmetric segregation of chromatids so that one daughter cell contains only the old intact DNA and the other carries chromatids composed exclusively of the newly synthesized DNA (Figure 1). This process would attenuate the accumulation of replication errors in the parental DNA strand.2,7 In the event that deleterious mutations are acquired during DNA replication, they would be transmitted to daughter cells, which may undergo senescence and apoptosis,8–11 having a reduced capacity to repair DNA damage.2,12,13 Moreover, telomeric shortening dictated by DNA synthesis would affect only partly the actual stem cells retaining the old DNA, and telomere attrition would be largely restricted to the newly synthesized strands and become templates in subsequent descendants.6,14 Figure 1. Nonrandom and random chromatid segregation. With asymmetric chromatid segregation, a dividing mother …

This review article addresses the controversy as to whether the adult heart possesses an intrinsic growth reserve. If myocyte renewal takes place in healthy and diseased organs, the reconstitution of the damaged tissue lost upon pathological insults might be achieved by enhancing a natural occurring process. Evidence in support of the old and new view of cardiac biology is critically discussed in an attempt to understand whether the heart is a static or dynamic organ. According to the traditional concept, the heart exerts its function until death of the organism with the same or lesser number of cells that are present at birth. This paradigm was challenged by documentation of the cell cycle activation and nuclear and cellular division in a subset of myocytes. These observations raised the important question of the origin of replicating myocytes. Several theories have been proposed and are presented in this review article. Newly formed myocytes may derive from a pre-existing pool of cells that has maintained the ability to divide. Alternatively, myocytes may be generated by activation and commitment of resident cardiac stem cells or by migration of progenitor cells from distant organs. In all cases, parenchymal cell turnover throughout lifespan results in a heterogeneous population consisting of young, adult, and senescent myocytes. With time, accumulation of old myocytes has detrimental effects on cardiac performance and may cause the development of an aging myopathy.

BACKGROUND Although progenitor cells have been described in distinct anatomical regions of the lung, description of resident stem cells has remained elusive. METHODS Surgical lung-tissue specimens were studied in situ to identify and characterize human lung stem cells. We defined their phenotype and functional properties in vitro and in vivo. RESULTS Human lungs contain undifferentiated human lung stem cells nested in niches in the distal airways. These cells are self-renewing, clonogenic, and multipotent in vitro. After injection into damaged mouse lung in vivo, human lung stem cells form human bronchioles, alveoli, and pulmonary vessels integrated structurally and functionally with the damaged organ. The formation of a chimeric lung was confirmed by detection of human transcripts for epithelial and vascular genes. In addition, the self-renewal and long-term proliferation of human lung stem cells was shown in serial-transplantation assays. CONCLUSIONS Human lungs contain identifiable stem cells. In animal models, these cells participate in tissue homeostasis and regeneration. They have the undemonstrated potential to promote tissue restoration in patients with lung disease. (Funded by the National Institutes of Health.).

Rationale: The ability of the human heart to regenerate large quantities of myocytes remains controversial, and the extent of myocyte renewal claimed by different laboratories varies from none to nearly 20% per year. Objective: To address this issue, we examined the percentage of myocytes, endothelial cells, and fibroblasts labeled by iododeoxyuridine in postmortem samples obtained from cancer patients who received the thymidine analog for therapeutic purposes. Additionally, the potential contribution of DNA repair, polyploidy, and cell fusion to the measurement of myocyte regeneration was determined. Methods and Results: The fraction of myocytes labeled by iododeoxyuridine ranged from 2.5% to 46%, and similar values were found in fibroblasts and endothelial cells. An average 22%, 20%, and 13% new myocytes, fibroblasts, and endothelial cells were generated per year, suggesting that the lifespan of these cells was approximately 4.5, 5, and 8 years, respectively. The newly formed cardiac cells showed a full...

Rationale: Chronic rejection, accelerated coronary atherosclerosis, myocardial infarction, and ischemic heart failure determine the unfavorable evolution of the transplanted heart in humans. Objective: Here we tested whether the pathological manifestations of the transplanted heart can be corrected partly by a strategy that implements the use of cardiac progenitor cells from the recipient to repopulate the donor heart with immunocompatible cardiomyocytes and coronary vessels. Methods and Results: A large number of cardiomyocytes and coronary vessels were created in a rather short period of time from the delivery, engraftment, and differentiation of cardiac progenitor cells from the recipient. A proportion of newly formed cardiomyocytes acquired adult characteristics and was integrated structurally and functionally within the transplant. Similarly, the regenerated arteries, arterioles, and capillaries were operative and contributed to the oxygenation of the chimeric myocardium. Attenuation in the extent of acute damage by repopulating cardiomyocytes and vessels decreased significantly the magnitude of myocardial scarring preserving partly the integrity of the donor heart. Conclusions: Our data suggest that tissue regeneration by differentiation of recipient cardiac progenitor cells restored a significant portion of the rejected donor myocardium. Ultimately, immunosuppressive therapy may be only partially required improving quality of life and lifespan of patients with cardiac transplantation.

Background— The striated preferentially expressed gene (Speg) generates 4 different isoforms through alternative promoter use and tissue-specific splicing. Depending on the cell type, Speg isoforms may serve as markers of striated or smooth muscle differentiation. Methods and Results— To elucidate function of Speg gene isoforms, we disrupted the Speg gene locus in mice by replacing common exons 8, 9, and 10 with a lacZ gene. β-Galactosidase activity was detected in cardiomyocytes of the developing heart starting at day 11.5 days post coitum (dpc). β-Galactosidase activity in other cell types, including vascular smooth muscle cells, did not begin until 18.5 dpc. In the developing heart, protein expression of only Spegα and Spegβ isoforms was present in cardiomyocytes. Homozygous Speg mutant hearts began to enlarge by 16.5 dpc, and by 18.5 dpc, they demonstrated dilation of right and left atria and ventricles. These cardiac abnormalities in the absence of Speg were associated with a cellular hypertrophic response, myofibril degeneration, and a marked decrease in cardiac function. Moreover, Speg mutant mice exhibited significant neonatal mortality, with increased death occurring by 2 days after birth. Conclusions— These findings demonstrate that mutation of the Speg locus leads to cardiac dysfunction and a phenotype consistent with a dilated cardiomyopathy.

Recent data indicate that the heart is a self-renewing organ and contains a pool of progenitor cells (PCs). According to the new paradigm, this resident population of multipotent undifferentiated cells gives rise to myocytes, endothelial cells, smooth muscle cells and fibroblasts. Understanding the function of cardiac PCs is critical for the implementation of these cells in the treatment of the diseased human heart. However, cardiac repair is an extremely complex phenomenon. Efficient myocardial regeneration requires restoration of segmental and focal areas of myocardial scarring, replacement of damaged coronary arteries, arterioles and capillaries, and substitution of hypertrophied poorly contracting myocytes with smaller better functioning parenchymal cells. To achieve these goals, the acquisition of a more profound knowledge of the biology of cardiac PCs cells and their fate following pathologic insults represents an essential need.

IGF-I rescues diabetic heart defects and oxidative stress, although the underlying mechanism of action remains poorly understood. This study was designed to delineate the beneficial effects of IGF-I with a focus on RhoA, Akt, and eNOS coupling. Echocardiography was performed in normal or diabetic Friend Virus-B type (FVB) and IGF-I transgenic mice. Cardiomyocyte contractile properties were evaluated using peak shortening (PS), time-to-90% relengthening (TR90), and intracellular Ca2+ rise and decay. Diabetes reduced fraction shortening, PS, and intracellular Ca2+; it increased chamber size, prolonged TR90, and intracellular Ca2+ decay. Levels of RhoA mRNA, active RhoA, and O2− were elevated, whereas nitric oxide (NO) levels were reduced in diabetes. Diabetes-induced O2− accumulation was ablated by the NO synthase (NOS) inhibitor nitro-l-arginine methyl ester (l-NAME), indicating endothelial NOS (eNOS) uncoupling, all of which except heart size were negated by IGF-I. The IGF-I-elicited beneficial effects were mimicked by the Rho kinase inhibitor Y27632 and BH4. Diabetes depressed expression of Kv1.2 and dihydrofolate reductase (DHFR), increased β-myosin heavy-chain expression, stimulated p38 MAPK, and reduced levels of total Akt and phosphorylated Akt/eNOS, all of which with the exception of myosin heavy chain were attenuated by IGF-I. In addition, Y27632 and the eNOS coupler folate abrogated glucose toxicity-induced PS decline, TR90 prolongation, while it increased O2− and decreased NO and Kv1.2 levels. The DHFR inhibitor methotrexate impaired myocyte function, NO/O2− balance, and rescued Y27632-induced cardiac protection. These results revealed that IGF-I benefits diabetic hearts via Rho inhibition and antagonism of diabetes-induced decrease in pAkt, eNOS uncoupling, and K+ channel expression.

Recent evidence would suggest that the heart is not a terminally differentiated organ and has the ability to regenerate itself under normal and pathophysiologic conditions. A major effort has been made to identify precursor cells that are capable of differentiating into cell lineages different from their organ of origin. Embryonic stem cells and bone marrow-derived cells (BMCs) have been studied and characterized, and BM precursor cells are currently being utilized as therapy in clinical trials of patients with heart failure of ischemic and nonischemic etiologies. Controversy remains, however, whether BMCs are the best cells to be used for replacement therapy. The existence of a cardiac stem cell (CSC) has also been described, which has the ability to generate new cardiac myocytes and blood vessels, raising the possibility of rebuilding a damaged heart with the organ's own precursor stem cell population. Animal studies have suggested such a possibility, and a clinical trial using CSCs is in progress. This monograph discusses our current understanding of myocardial regeneration and the roles that endogenous and exogenous stem cells may have in the future therapy of cardiovascular disease.

The serine-threonine kinases Pim-1 and Akt regulate cellular proliferation and survival. Although Akt is known to be a crucial signaling protein in the myocardium, the role of Pim-1 has been overlooked. Pim-1 expression in the myocardium of mice decreased during postnatal development, re-emerged after acute pathological injury in mice and was increased in failing hearts of both mice and humans. Cardioprotective stimuli associated with Akt activation induced Pim-1 expression, but compensatory increases in Akt abundance and phosphorylation after pathological injury by infarction or pressure overload did not protect the myocardium in Pim-1-deficient mice. Transgenic expression of Pim-1 in the myocardium protected mice from infarction injury, and Pim-1 expression inhibited cardiomyocyte apoptosis with concomitant increases in Bcl-2 and Bcl-X(L) protein levels, as well as in Bad phosphorylation levels. Relative to nontransgenic controls, calcium dynamics were significantly enhanced in Pim-1-overexpressing transgenic hearts, associated with increased expression of SERCA2a, and were depressed in Pim-1-deficient hearts. Collectively, these data suggest that Pim-1 is a crucial facet of cardioprotection downstream of Akt.

A problem in need of resolution concerns the origin of cardiac progenitor cells and the mechanisms by which these cells are preserved within the cardiac niches. This might be accomplished by migration of progenitor cells from the bone marrow to the myocardium. Alternatively, the progenitor cell compartment in the heart may be maintained by asymmetric division of resident cells. These two possibilities are not mutually exclusive and both exogenous and endogenous progenitor cells may contribute to cardiac homeostasis.

The recognition that the adult heart continuously renews its myocyte compartment raises the possibility that the age and lifespan of myocytes does not coincide with the age and lifespan of the organ and organism. If this were the case, myocyte turnover would result at any age in a myocardium composed by a heterogeneous population of parenchymal cells which are structurally integrated but may contribute differently to myocardial performance. To test this hypothesis, left ventricular myocytes were isolated from mice at 3 months of age and the contractile, electrical, and calcium cycling characteristics of these cells were determined together with the expression of the senescence-associated protein p16 INK4a and telomere length. The heart was characterized by the coexistence of young, aged, and senescent myocytes. Old nonreplicating, p16 INK4a -positive, hypertrophied myocytes with severe telomeric shortening were present together with young, dividing, p16 INK4a -negative, small myocytes with long telomeres. A class of myocytes with intermediate properties was also found. Physiologically, evidence was obtained in favor of the critical role that action potential (AP) duration and I CaL play in potentiating Ca 2+ cycling and the mechanical behavior of young myocytes or in decreasing Ca 2+ transients and the performance of senescent hypertrophied cells. The characteristics of the AP appeared to be modulated by the transient outward K + current I to which was influenced by the different expression of the K + channels subunits. Collectively, these observations at the physiological and structural cellular level document that by necessity the heart has to constantly repopulate its myocyte compartment to replace senescent poorly contracting myocytes with younger more efficient cells. Thus, cardiac homeostasis and myocyte turnover regulate cardiac function.

If I can stop one heart from breaking — -Emily Dickinson 1 In the Ode to Broken Things , Pablo Neruda tenders a powerful metaphor of existence.2 It is not difficult to recognize the unwinding of human life in the broken clock that was once “the secret thread of our weeks” and now, with its “blue guts” exposed and its “wide heart unsprung,” is the symbol of agony and death. The beginning and ending of life are embraced in a poem about simple and ordinary things that accompany human beings throughout life and then are lost, together with the feelings and significance that were assigned to them. All things break, even the heart. What to do for broken things and broken hearts? The poet suggests collecting our treasures and sinking them in the ocean with the hope that the “long labor of its tides” may give back wholeness to the fragments. While we wait for the sea to reveal its strength and miraculous effects, the discovery that stem cells repair broken organs projects a more hopeful view of medicine and the way it is practiced. Article p 896 Soon after the first experimental evidence that bone marrow cells (BMCs) induce cardiac repair in the postinfarcted heart,3 unfractionated mononuclear BMCs and CD34-positive cells were given to patients affected by acute myocardial infarction or chronic ischemic heart failure.4,5 Results accumulated so far have documented the feasibility of this therapeutic approach with indications of potential beneficial effects on cardiac function and critical clinical end points. Although the mechanisms by which BMCs improve ventricular performance in humans are currently unknown, experimental evidence favors cardiac regeneration and the production of paracrine factors by these cells.6 New myocytes and coronary vessels are formed by transdifferentiation of the delivered cells, a process that …

This review discusses the current controversy about the role that endogenous and exogenous progenitor cells have in cardiac homeostasis and myocardial regeneration following injury. Although great enthusiasm was created by the possibility of reconstituting the damaged heart, the opponents of this new concept of cardiac biology have interpreted most of the findings supporting this possibility as the product of technical artifacts. This article challenges this established, static view of cardiac growth and favors the notion that the mammalian heart has the inherent ability to replace its cardiomyocytes through the activation of a pool of resident primitive cells or the administration of hematopoietic stem cells.

The preservation of myocyte number and cardiac mass throughout life is dependent on the balance between cell death and cell division. Rapidly emerging evidence indicates that new myocytes can be formed through the activation and differentiation of resident cardiac progenitor cells. The critical issue is the identification of mechanisms that define the aging of cardiac progenitor cells and, ultimately, their inability to replace dying myocytes. The most reliable marker of cellular senescence is the modification of the telomere-telomerase axis, together with the expression of the cell cycle inhibitors p16INK4a and p53. Cellular senescence is characterized by biochemical events that occur within the cell. In this regard, one of the most relevant processes is represented by repeated oxidative stress that may evolve into the activation of the cell death program or result in the development of a senescent phenotype. Thus, the modulation of telomerase activity and the control of telomeric length, together with the attenuation of the formation of reactive oxygen species, may represent important therapeutic tools in regenerative medicine and in prevention of aging and diabetic cardiomyopathies.

Activation of Akt is associated with enhanced cell cycling and cellular proliferation in nonmyocytes, but this effect of nuclear Akt accumulation has not been explored in the context of the myocardium. Cardiac-specific expression of nuclear-targeted Akt (Akt/nuc) in transgenics prolongs postnatal cell cycling as evidenced by increased numbers of Ki67 + cardiomyocytes at 2 to 3 weeks after birth. Similarly, nuclear-targeting of Akt promotes expansion of the presumptive cardiac progenitor cell population as assessed by immunolabeling for c-kit in combination with myocyte-specific markers Nkx 2.5 or MEF 2C. Increases in pro-proliferative cytokines, including tumor-necrosis superfamily 8, interleukin-17e, and hepatocyte growth factor, were found in nuclear-targeted Akt myocardial samples. Concurrent signaling mediated by paracrine factors downstream of Akt/nuc expression may be responsible for phenotypic effects of nuclear-targeted Akt in the myocardium, including enhanced cell proliferation and expansion of the stem cell population.

The serine/threonine kinase Akt regulates cellular survival, proliferation, gene transcription, protein translation, metabolism, and differentiation. Although Akt substrates are found throughout the cell, activated Akt normally accumulates in the nucleus, suggesting that biologically relevant targets are located there. Consequences of nuclear Akt signaling in cardiomyocytes were explored by using nuclear-targeted Akt (Akt-nuc). Accumulation of Akt-nuc did not provoke hypertrophy, unlike constitutively activated Akt. Instead, Akt-nuc inhibited hypertrophy concurrent with increased atrial natriuretic peptide (ANP) expression that depended upon phosphatidylinositol-3 kinase activity. Akt-nuc antihypertrophic effects were blocked by inhibition of either guanylyl cyclase A receptor or cyclic guanosine monophosphate-dependent protein kinase in cultured cardiomyocytes. Corroborating evidence showed blunted acute hypertrophic remodeling in Akt-nuc transgenic mice after transverse aortic constriction coincident with higher ANP expression and smaller myocyte volume. In addition, Akt-nuc expression improved systolic function and survival in the chronic phase of transverse aortic constriction-induced hypertrophy. Thus, Akt-nuc antagonizes certain aspects of hypertrophy through autocrine/paracrine stimulation of a phosphatidylinositol-3 kinase-dependent signaling cascade that promotes ANP expression, resulting in a unique combination of prosurvival coupled with antihypertrophic signaling.

In the last decade, apoptosis has gained recognition as an important event determining the structure and function of the myocardium. The purpose of this review is to provide insights into the mechanisms controlling programmed cell death of myocytes. In this regard, the role of the local renin-angiotensin system and oxidative stress is emphasized. Moreover, the effect of cell death on ventricular remodeling and performance is analyzed. The impact of the identification of apoptosis in the heart on the current view of myocardium as a dynamic organ is discussed.

Annarosa Leri1, Toru Hosoda1, Jan Kajstura1 & Piero Anversa2† †Author for correspondence 1Cardiovascular Research Institute, Department of Medicine, New York Medical College, Valhalla, New York, NY 10595, USA 2Cardiovascular Research Institute, Department of Medicine, Vosburgh Pavilion, Room 302, New York Medical College, Valhalla, New York, NY 10595, USA Tel.: +1 914 594 4168; Fax: +1 914 594 4406; E-mail: piero_anversa@ nymc.edu ‘The bizarre behavior of hematopoietic stem cells has surprised and distressed many of us; they disobey the dogma of embryonic specification and with elegant and plastic arabesques undergo unexpected metamorphoses.’

This review questions the old paradigm that describes the heart as a post-mitotic organ and introduces the notion of the heart as a self-renewing organ regulated by a compartment of multipotent cardiac stem cells (CSCs) capable of regenerating myocytes and coronary vessels throughout life. Because of this dramatic change in cardiac biology, the objective is to provide an alternative perspective of the aging process of the heart and stimulate research in an area that pertains to all of us without exception. The recent explosion of the field of stem cell biology, with the recognition that the possibility exists for extrinsic and intrinsic regeneration of myocytes and coronary vessels, necessitates reevaluation of cardiac homeostasis and myocardial aging. From birth to senescence, the mammalian heart is composed of non-dividing and dividing cells. Loss of telomeric DNA is minimal in fetal and neonatal myocardium but rather significant in the senescent heart. Aging affects the growth and differentiation potential of CSCs interfering not only with their ability to sustain physiological cell turnover but also with their capacity to adapt to increases in pressure and volume loads. The recognition of factors enhancing the activation of the CSC pool, their mobilization, and translocation, however, suggests that the detrimental effects of aging on the heart might be prevented or reversed by local stimulation of CSCs or the intramyocardial delivery of CSCs following their expansion and rejuvenation in vitro. CSC therapy may become, perhaps, a novel strategy for the devastating problem of heart failure in the old population.

The purpose of this study was to determine whether the heart in large mammals contains cardiac progenitor cells that regulate organ homeostasis and regenerate dead myocardium after infarction. We report that the dog heart possesses a cardiac stem cell pool characterized by undifferentiated cells that are self-renewing, clonogenic, and multipotent. These clonogenic cells and early committed progeny possess a hepatocyte growth factor (HGF)–c-Met and an insulin-like growth factor 1 (IGF-1)-IGF-1 receptor system that can be activated to induce their migration, proliferation, and survival. Therefore, myocardial infarction was induced in chronically instrumented dogs implanted with sonomicrometric crystals in the region of the left ventricular wall supplied by the occluded left anterior descending coronary artery. After infarction, HGF and IGF-1 were injected intramyocardially to stimulate resident cardiac progenitor cells. This intervention led to the formation of myocytes and coronary vessels within the infarct. Newly generated myocytes expressed nuclear and cytoplasmic proteins specific of cardiomyocytes: MEF2C was detected in the nucleus, whereas α-sarcomeric actin, cardiac myosin heavy chain, troponin I, and α-actinin were identified in the cytoplasm. Connexin 43 and N-cadherin were also present. Myocardial reconstitution resulted in a marked recovery of contractile performance of the infarcted heart. In conclusion, the activation of resident primitive cells in the damaged dog heart can promote a significant restoration of dead tissue, which is paralleled by a progressive improvement in cardiac function. These results suggest that strategies capable of activating the growth reserve of the myocardium may be important in cardiac repair after ischemic injury.

Stem cell transdifferentiation in the adult organism is the most common and questioned mechanism of growth and repair. Recent data suggest that adult stem cells are capable of generating mature cells beyond their own tissue boundaries, a process called developmental plasticity. To date, the most versatile cell discovered is the bone marrow progenitor cell.

Recent studies in mice have challenged the ability of bone marrow cells (BMCs) to differentiate into myocytes and coronary vessels. The claim has also been made that BMCs acquire a cell phenotype different from the blood lineages only by fusing with resident cells. Technical problems exist in the induction of myocardial infarction and the successful injection of BMCs in the mouse heart. Similarly, the accurate analysis of the cell populations implicated in the regeneration of the dead tissue is complex and these factors together may account for the negative findings. In this study, we have implemented a simple protocol that can easily be reproduced and have reevaluated whether injection of BMCs restores the infarcted myocardium in mice and whether cell fusion is involved in tissue reconstitution. For this purpose, c-kit–positive BMCs were obtained from male transgenic mice expressing enhanced green fluorescence protein (EGFP). EGFP and the Y-chromosome were used as markers of the progeny of the transplanted cells in the recipient heart. By this approach, we have demonstrated that BMCs, when properly administrated in the infarcted heart, efficiently differentiate into myocytes and coronary vessels with no detectable differentiation into hemopoietic lineages. However, BMCs have no apparent paracrine effect on the growth behavior of the surviving myocardium. Within the infarct, in 10 days, nearly 4.5 million biochemically and morphologically differentiated myocytes together with coronary arterioles and capillary structures were generated independently of cell fusion. In conclusion, BMCs adopt the cardiac cell lineages and have an important therapeutic impact on ischemic heart failure.

Heart failure is associated with death of cardiomyocytes leading to loss of contractility. Previous studies using membrane-targeted Akt (myristolated-Akt), an enzyme involved in antiapoptotic signaling, showed inhibition of cell death and prevention of pathogenesis induced by cardiomyopathic stimuli. However, recent studies by our group have found accumulation of activated Akt in the nucleus, suggesting that biologically relevant target(s) of Akt activity may be located there. To test this hypothesis, a targeted Akt construct was created to determine the antiapoptotic action of nuclear Akt accumulation. Nuclear localization of the adenovirally encoded Akt construct was confirmed by confocal microscopy. Cardiomyocytes expressing nuclear-targeted Akt showed no evidence of morphological remodeling such as altered myofibril density or hypertrophy. Nuclear-targeted Akt significantly elevated levels of phospho-Akt and kinase activity and inhibited apoptosis as effectively as myristolated-Akt in hypoxia-induced cell death. Transgenic overexpression of nuclear-targeted Akt did not result in hypertrophic remodeling, altered cardiomyocyte DNA content or nucleation, or enhanced phosphorylation of typical cytoplasmic Akt substrates, yet transgenic hearts were protected from ischemia-reperfusion injury. Gene array analyses demonstrated changes in the transcriptional profile of Akt/nuc hearts compared with nontransgenic controls distinct from prior characterizations of Akt expression in transgenic hearts. Collectively, these experiments show that targeting of Akt to the nucleus mediates inhibition of apoptosis without hypertrophic remodeling, opening new possibilities for therapeutic applications of nuclear-targeted Akt to inhibit cell death associated with heart disease.

In the last few years, several experimental studies have used stem cells of different sources to reconstitute damaged tissues.1 The brain and the heart have been the most investigated organs because of the long-standing view of the lack of regenerating potential of neurons and myocytes.2,3 Bone marrow stem cells (BMSCs) have been reported capable of transdifferentiating in various cell lineages distinct from the site of origin4 and, because of this property, they may constitute a new form of cellular therapy. Neuronal and myocardial growth mediated by bone marrow cells (BMCs) has been demonstrated, but these results have been challenged5,6 and the issue of BMSC transdifferentiation has become highly controversial. Heated debates at scientific meetings, letters in high-profile journals, and reports with contradicting observations have raised questions on the plasticity of BMSCs.5–7 If negative results would be more cautiously interpreted instead of being blown out of context, it is likely that the actual role that adult stem cells play in the repair of tissues and organs would be better understood and appreciated. This is particularly relevant when negative data are dropped as “valid” statements from the podium and are quoted before they undergo peer review and publication.6 A good example of the opposite approach is found in the study of Chen and colleagues8 in this issue of Circulation Research . The authors have utilized, among other sophisticated techniques, confocal microscopy to identify and characterize an important new function of human pluripotent adult mesenchymal BMSCs. In this report, an unequivocal demonstration was obtained on the ability of these cells to deliver vascular endothelial growth factor (VEGF) to an ischemic region of the brain. VEGF accumulation coupled with endogenous activation of endothelial cells and VEGF synthesis promoted vessel formation after stroke. BMSCs via VEGF secretion acted …

The accepted paradigm considers the adult mammalian heart as a postmitotic organ, which possesses a relatively constant number of myocytes from shortly after birth to adulthood and senescence. This notion is questioned by the demonstration that although most adult myocytes are terminally differentiated, there is a small and continuously renewed subpopulation of cycling myocytes produced by the differentiation of cardiac stem-like cells. Myocyte death and myocyte regeneration are introduced as major determinants of cardiac homeostasis and alterations of ventricular anatomy and function in physiological and pathological states. The possibility of reconstituting dead myocardium by stem-like cells is advanced and proposed as a major area of future research.

Cardiac failure is a frequent cause of death in the aging human population. Telomere attrition occurs with age, and is proposed to be causal for the aging process. To determine whether telomere shortening leads to a cardiac phenotype, we studied heart function in the telomerase knockout mouse, Terc–/–. We studied Terc–/– mice at the second, G2, and fifth, G5, generation. Telomere shortening in G2 and G5 Terc–/– mice was coupled with attenuation in cardiac myocyte proliferation, increased apoptosis and cardiac myocyte hypertrophy. On a single-cell basis, telomere shortening was coincidental with increased expression of p53, indicating the presence of dysfunctional telomeres in cardiac myocytes from G5 Terc–/– mice. The impairment in cell division, the enhanced cardiac myocyte death and cellular hypertrophy, are concomitant with ventricular dilation, thinning of the wall and cardiac dysfunction. Thus, inhibition of cardiac myocyte replication provoked by telomere shortening, results in de-compensated eccentric hypertrophy and heart failure in mice. Telomere shortening with age could also contribute to cardiac failure in humans, opening the possibility for new therapies.

Improvement in the methodological approach to the analysis of the myocardium has provided clear evidence of cardiac myocyte proliferation, questioning the general belief that the growth of the adult heart under physiological and pathological conditions can occur only by cellular hypertrophy. Myocyte regeneration contributes via myocyte death to the physiological turnover of myocytes and via myocyte hypertrophy to cardiac remodeling. Several questions, however, remain to be answered. Among them, it is still unknown whether myocyte multiplication exerts a positive and/or negative effect on ventricular anatomy and cardiac function. The addition of newly generated myocytes leads to cavitary dilation with relative thinning of the wall. Conversely, myocyte proliferation, characterized by the parallel addition of cells, can be expected to increase wall thickness, decrease chamber size, and ameliorate cardiac performance.

To test the hypothesis that early interventional treatment with insulin-like growth factor-1 (IGF-1) alleviates subsequent development of dilated cardiomyopathy, cardiac-specific IGF-1 expression was introduced by selective cross-breeding into a transgenic mouse model of heart failure that displays phenotypic characteristics of severe dilation. Hemodynamic, structural, and cellular parameters of the heart were compared between nontransgenic, tropomodulin-overexpressing cardiomyopathic, and the hybrid tropomodulin/IGF-1-overexpressing mice. Beneficial effects of IGF-1 were apparent by multiple indices of cardiac structure and function, including normalization of heart mass, anatomy, hemodynamics, and apoptosis. IGF-1 expression also acted as a proliferative stimulus as evidenced by calculated increases in myocyte number as well as expression of Ki67, a nuclear marker of cellular replication. Cellular analyses revealed that IGF-1 inhibited characteristic cardiomyocyte elongation in dilated hearts and restored calcium dynamics comparable to that observed in normal cells. Collectively, these results provide novel information regarding the ability of IGF-1 to inhibit progression of cardiomyopathic disease in a defined model system and suggest that heart failure may benefit from early interventional IGF-1 treatment.

Little is known about the function of inositol 1,4,5-trisphosphate receptors (IP3Rs) in the adult heart experimentally. Moreover, whether these Ca(2+) release channels are present and play a critical role in human cardiomyocytes remains to be defined. IP3Rs may be activated after Gαq-protein-coupled receptor stimulation, affecting Ca(2+) cycling, enhancing myocyte performance, and potentially favoring an increase in the incidence of arrhythmias.IP3R function was determined in human left ventricular myocytes, and this analysis was integrated with assays in mouse myocytes to identify the mechanisms by which IP3Rs influence the electric and mechanical properties of the myocardium. We report that IP3Rs are expressed and operative in human left ventricular myocytes. After Gαq-protein-coupled receptor activation, Ca(2+) mobilized from the sarcoplasmic reticulum via IP3Rs contributes to the decrease in resting membrane potential, prolongation of the action potential, and occurrence of early afterdepolarizations. Ca(2+) transient amplitude and cell shortening are enhanced, and extrasystolic and dysregulated Ca(2+) elevations and contractions become apparent. These alterations in the electromechanical behavior of human cardiomyocytes are coupled with increased isometric twitch of the myocardium and arrhythmic events, suggesting that Gαq-protein-coupled receptor activation provides inotropic reserve, which is hampered by electric instability and contractile abnormalities. Additionally, our findings support the notion that increases in Ca(2+) load by IP3Rs promote Ca(2+) extrusion by forward-mode Na(+)/Ca(2+) exchange, an important mechanism of arrhythmic events.The Gαq-protein/coupled receptor/IP3R axis modulates the electromechanical properties of the human myocardium and its propensity to develop arrhythmias.

The role and even the existence of myocyte proliferation in the adult heart remain controversial. Documentation of cell cycle regulators, DNA synthesis, and mitotic images has not modified the view that myocardial growth can only occur from hypertrophy of an irreplaceable population of differentiated myocytes. To improve understanding the biology of the heart and obtain supportive evidence of myocyte replication, three indices of cell proliferation were analyzed in dogs affected by a progressive deterioration of cardiac performance and dilated cardiomyopathy. The magnitude of cycling myocytes was evaluated by the expression of Ki67 in nuclei. Ki67 labeling of left ventricular myocytes increased 5-fold, 12-fold, and 17-fold with the onset of moderate and severe ventricular dysfunction and overt failure, respectively. Telomerase activity in vivo is present only in multiplying cells; this enzyme increased 2.4-fold and 3.1-fold in the decompensated heart, preserving telomeric length in myocytes. The contribution of cycling myocytes to telomerase activity was determined by the colocalization of Ki67 and telomerase in myocyte nuclei. More than 50% of Ki67-positive cells expressed telomerase in the overloaded myocardium, suggesting that these myocytes were the morphological counterpart of the biochemical assay of enzyme activity. Moreover, we report that 20–30% of canine myocytes were telomerase competent, and this value was not changed by cardiac failure. In conclusion, the enhanced expression of Ki67 and telomerase activity, in combination with Ki67-telomerase labeling of myocyte nuclei, support the notion that myocyte proliferation contributes to cardiac hypertrophy of the diseased heart.

Abstract —Cardiovascular disease risk is higher in men than women, but the basis for this discrepancy remains controversial. Estrogenic stimulation of the myocardium or isolated cardiomyocytes has been purported to exert multiple beneficial effects associated with inhibition of maladaptive responses to pathogenic insults. This report describes a significant difference between the sexes in myocardial activation of Akt, a protein kinase that regulates a broad range of physiological responses including metabolism, gene transcription, and cell survival. We find that young women possess higher levels of nuclear-localized phospho-Akt 473 relative to comparably aged men or postmenopausal women. Both localization of phospho-Akt 473 in myocardial nuclei of sexually mature female mice versus males and Akt kinase activity in nuclear extracts of hearts from female mice versus males are elevated. Cytosolic localization of phospho-forkhead, a downstream nuclear target of Akt, is also increased in female relative to male mice, suggesting a potential mechanism for cardioprotective nuclear signaling resulting from Akt activation. Phospho-Akt 473 levels and localization at cardiac nuclei are similarly increased in transgenic mice with myocardium-specific expression of insulin-like growth factor I, a proven stimulus for Akt activation. Phospho-Akt 473 is also localized to the nucleus of cultured cardiomyocytes after exposure to 17β-estradiol or genistein (a phytoestrogen in soy protein–based diets), and neonatal exposure of litters to genistein elevated nuclear phospho-Akt 473 localization. The activation of Akt in a gender-dependent manner may help explain differences observed in cardiovascular disease risk between the sexes and supports the potential beneficial effects of estrogenic stimulation.

Abstract —Ventricular pacing leads to a dilated myopathy in which cell death and myocyte hypertrophy predominate. Because angiotensin II (Ang II) stimulates myocyte growth and triggers apoptosis, we tested whether canine myocytes express the components of the renin-angiotensin system (RAS) and whether the local RAS is upregulated with heart failure. p53 modulates transcription of angiotensinogen (Aogen) and AT 1 receptors in myocytes, raising the possibility that enhanced p53 function in the decompensated heart potentiates Ang II synthesis and Ang II–mediated responses. Therefore, the presence of mRNA transcripts for Aogen, renin, angiotensin-converting enzyme, chymase, and AT 1 and AT 2 receptors was evaluated by reverse transcriptase–polymerase chain reaction in myocytes. Changes in the protein expression of these genes were then determined by Western blot in myocytes from control dogs and dogs affected by congestive heart failure. p53 binding to the promoter of Aogen and AT 1 receptor was also determined. Ang II in myocytes was measured by ELISA and by immunocytochemistry and confocal microscopy. Myocytes expressed mRNAs for all the constituents of RAS, and heart failure was characterized by increased p53 DNA binding to Aogen and AT 1 . Additionally, protein levels of Aogen, renin, cathepsin D, angiotensin-converting enzyme, and AT 1 were markedly increased in paced myocytes. Conversely, chymase and AT 2 proteins were not altered. Ang II quantity and labeling of myocytes increased significantly with cardiac decompensation. In conclusion, dog myocytes synthesize Ang II, and activation of p53 function with ventricular pacing upregulates the myocyte RAS and the generation and secretion of Ang II. Ang II may promote myocyte growth and death, contributing to the development of heart failure.

To determine whether stretch-induced activation of p53 is necessary for the up-regulation of the local renin-angiotensin system and angiotensin II (Ang II)-induced apoptosis, ventricular myocytes were infected with an adenoviral vector carrying mutated p53, Adp53m, before 12 hours of stretch. Noninfected myocytes and myocytes infected with AdLacZ served as controls. Stretching of Adp53m-infected myocytes prevented stimulation of p53 function that conditioned the expression of p53-dependent genes; quantity of angiotensinogen (Aogen), AT(1), and Bax decreased, whereas Bcl-2 increased. Ang II generation was not enhanced by stretch. Conversely, stretch produced opposite changes in noninfected and AdLacZ-infected myocytes: Aogen increased twofold, AT(1) increased 2. 1-fold, Bax increased 2.5-fold, and Ang II increased 2.4-fold. These responses were coupled with 4.5-fold up-regulation of wild-type p53. Stretch elicited comparable adaptations in p53-independent genes, in the presence or absence of mutated p53; renin increased threefold, angiotensin-converting enzyme increased ninefold, and AT(2) increased 1.7-fold. Infection with Adp53m inhibited myocyte apoptosis after stretch. Conversely, stretch increased apoptosis by 6.2-fold in myocytes with elevated endogenous wild-type p53. Thus, a competitor of p53 function interfered with both stretch-induced Ang II formation and apoptosis, indicating that p53 is a major modulator of myocyte renin-angiotensin system and cell survival after mechanical deformation.

To determine whether myocyte death and angiotensin II (AT II) formation are implicated in the development of diabetic cardiomyopathy, rats were injected with streptozotocin, and apoptosis and necrosis were measured at 3, 10, and 28 days. Expression of the components of the renin-angiotensin system (RAS) and AT II levels were assessed at 3 days. The percentage of AT II-labeled myocytes and the number and distribution of AT II sites in myocytes were measured at 3 and 10 days. The effects of AT1 blockade on local RAS and cell death were examined at 3 days. Diabetes was characterized by myocyte apoptosis that peaked at 3 days and decreased at 10 and 28 days, in spite of high concentrations of blood glucose. Cell necrosis was absent throughout. Angiotensinogen, renin, and AT1 receptor increased in myocytes from diabetic rat hearts, while angiotensin-converting enzyme and AT2 remained constant. AT II quantity increased severalfold, as did the fraction of AT II positive cells and the number of AT II sites per myocyte. However, AT II labeling decreased at 10 days, which paralleled the reduction in myocyte death. AT1 antagonist inhibited upregulation of this receptor and angiotensinogen, which prevented AT II synthesis and myocyte death at their peaks with diabetes. An aggregate 30% myocyte loss and a 14% increase in the volume of viable cells were found in diabetic rats at 28 days. Thus diabetic cardiomyopathy may be viewed as an AT II-dependent process in which that peptide plays a critical role in myocyte death and hypertrophy.

To determine whether adult cardiac myocytes are capable of multiple divisions and whether this form of growth is restricted to a subpopulation of cells that retain this capacity with age, telomere lengths were measured in myocyte nuclei isolated from the left ventricle of fetal and neonatal Fischer 344 rats and rats at 4, 12, and 27 months after birth. Two independent methodologies were used for this analysis: laser scanning cytometer and confocal microscopy. In each case, fluorescence intensity of a peptide nucleic acid probe specific for telomeric sequence was evaluated. The two techniques yielded comparable results. Telomeric shortening increased with age in a subgroup of myocytes that constituted 16% of the entire cell population. In the remaining nondividing cells, progressive accumulation of a senescent associated nuclear protein, p16(INK4), was evidenced. In conclusion, a significant fraction of myocytes divides repeatedly from birth to senescence, counteracting the continuous death of cells in the aging mammalian rat heart.

Telomerase replaces telomeric repeat DNA lost during the cell cycle, restoring telomere length. This enzyme is present only during cell replication and its activity reflects the extent of proliferation. Whether cardiac myocytes are terminally differentiated cells is still a highly controversial issue, and the possibility of myocyte division is frequently rejected. On this basis, telomerase was measured in pure preparations of myocytes, isolated from rats throughout their lifespan. Fetal and neonatal rat myocytes were used as positive control cells. Contrary to expectation, the authors report that telomerase activity was detectable in pure preparations of young adult, fully mature adult, and senescent ventricular myocytes, defeating the dogma that this cell population is permanent and irreplaceable. Aging decreased 31% telomerase activity in male myocytes. An opposite effect occurred in female myocytes in which this enzyme increased 72%. This differential adaptation between the two genders in the rat model may be relevant to observations in humans; myocyte loss occurs in men as a function of age, whereas myocyte number is preserved in women. The greater growth potential of female myocytes may be critical for the longer lifespan and decreased incidence of heart failure in women.

Abstract —Coronary artery narrowing (CAN) induces tissue injury, which may involve myocyte necrosis and apoptosis. Insulin-like growth factor (IGF)–1 may counteract cell death, modifying the detrimental effects of myocardial ischemia. On this basis, CAN was produced in female FVB. Igf +/– mice and nontransgenic littermates, and the animals were euthanized 7 days later. CAN consisted of an 82% reduction in the vessel luminal cross-sectional area in both groups of mice. Severe left ventricular dysfunction was present in CAN nontransgenic and transgenic mice, but heart and left ventricular weights increased more in littermates than in FVB. Igf +/– mice. Similarly, the changes in chamber volume and diastolic wall stress were greater in nontransgenic mice. Subacute tissue injury, represented by foci of replacement fibrosis, was 2.6-fold higher in CAN littermates than in FVB. Igf +/– mice. Ongoing myocyte necrosis was 5-fold greater in nontransgenic mice, whereas apoptosis was low and did not differ in the 2 groups of mice. In CAN nontransgenic mice, myocyte necrosis was 12-fold more frequent than apoptosis but, in CAN transgenic mice, these 2 types of cell death were comparable. α-Myosin and β-myosin isoform mRNAs were affected by CAN, but α-myosin mRNA was reduced more in nontransgenic mice. In conclusion, myocyte necrosis and replacement fibrosis are the prevailing forms of myocardial damage induced by CAN. Constitutive overexpression of IGF-1 attenuates myocyte necrosis and tissue injury, having no effect on cell apoptosis. These factors limit ventricular dilation, myocardial loading, cardiac hypertrophy, and alterations in α- and β-myosin isoform expression.

Abstract —Constitutive overexpression of insulin-like growth factor-1 (IGF-1) in myocytes protects them from apoptosis and interferes with myocyte hypertrophy in the normal and pathological heart. Conversely, angiotensin II (Ang II) triggers cell death and promotes myocyte hypertrophy. Moreover, activation of p53 upregulates the cellular renin-angiotensin system (RAS). Therefore, IGF-1 overexpression in FVB. Igf +/− mice may downregulate the local RAS through the attenuation of p53 and p53-inducible genes. On this basis, p53 DNA binding activity to angiotensinogen (Aogen), bax, and the AT 1 receptor was determined in left ventricular myocytes from FVB. Igf −/− and FVB. Igf +/− mice. The quantity of Bax, Bcl-2, Aogen, and AT 1 receptor in these cells was evaluated. The presence of Mdm2-p53 complexes was also established. Finally, Ang II levels in myocytes were measured. Upregulation of IGF-1 in myocytes was associated with a protein-to-protein interaction between Mdm2 and p53, which attenuated p53 transcriptional activity for bax, Aogen, and AT 1 receptor. Similarly, the amount of Bax, Aogen, and AT 1 receptor proteins in these cells decreased. In contrast, the expression of Bcl-2 remained constant. The downregulation of Aogen in myocytes from FVB. Igf +/− mice was characterized by a reduction in Ang II. In conclusion, IGF-1 negatively influences the myocyte RAS through the upregulation of Mdm2 and its binding to p53. This may represent the molecular mechanism responsible for the effects of IGF-1 on cell viability and myocyte hypertrophy in the nonpathological and pathological heart in vivo.

Insulin-like growth factor (IGF)-1 inhibits apoptosis, but its mechanism is unknown. Myocyte stretching activates p53 and p53-dependent genes, leading to the formation of angiotensin II (Ang II) and apoptosis. Therefore, this in vitro system was used to determine whether IGF-1 interfered with p53 function and the local renin-angiotensin system (RAS), decreasing stretch-induced cell death. A single dose of 200 ng/ml IGF-1 at the time of stretching decreased myocyte apoptosis 43% and 61% at 6 and 20 hours. Ang II concentration was reduced 52% at 20 hours. Additionally, p53 DNA binding to angiotensinogen (Aogen), AT 1 receptor, and Bax was markedly down-regulated by IGF-1 via the induction of Mdm2 and the formation of Mdm2-p53 complexes. Concurrently, the quantity of p53, Aogen, renin, AT 1 receptor, and Bax was reduced in stretched myocytes exposed to IGF-1. Conversely, Bcl-2 and the Bcl-2-to-Bax protein ratio increased. The effects of IGF-1 on cell death, Ang II synthesis, and Bax protein were the consequence of Mdm2-induced down-regulation of p53 function. In conclusion, the anti-apoptotic impact of IGF-1 on stretched myocytes was mediated by its capacity to depress p53 transcriptional activity, which limited Ang II formation and attenuated the susceptibility of myocytes to trigger their endogenous cell death pathway.

Primary cultures of various cell types contain dividing and nondividing cells.1 2 However, nondividing cells can rest in the G phase and reenter the cell cycle on stimulation3 4 5 or become terminally differentiated and die without dividing.6 This third category of cells is not dormant in G and cannot reenter the cell cycle.6 For example, such a phenomenon is present in vivo at the tip of the villi of the small intestine,7 in the auditory hair cells of the ear,8 and in the upper layers of the epidermis.9 In contrast, hepatocytes in vivo are in a G state,10 11 12 and after partial hepatectomy or severe injury, liver regeneration is accomplished by proliferation of mature hepatocytes as well as biliary epithelial cells and fenestrated endothelial cells.12 The reconstitution of liver mass is not dependent on a reserve of stem cells, which condition regeneration of bone marrow13 and skin.14 Multipotential cells have also been identified in the central nervous system, but their ability to grow, differentiate, and ultimately survive has been problematic.15 In skeletal muscle, satellite cells can be stimulated to proliferate and develop into mature myocytes, representing an additional form of tissue regeneration.16 17 18 These mechanisms of cellular growth have not been considered possible in adult cardiac myocytes, and the concept was advanced that no proliferation of ventricular muscle cells occurs once cell division has ceased, shortly after birth in the mammalian heart.19 The dogma was introduced that adult cardiac myocytes are terminally differentiated cells and, therefore, cannot be recalled into the cell cycle.20 21 These cells are not in G, cannot be triggered into the proliferative phase, but can perform their physiological functions, undergo cellular hypertrophy, and …