Abstract-To determine whether cellular aging leads to a cardiomyopathy and heart failure, markers of cellular senescence, cell death, telomerase activity, telomere integrity, and cell regeneration were measured in myocytes of aging wild-type mice (WT). These parameters were similarly studied in insulin-like growth factor-1 (IGF-1) transgenic mice (TG) because IGF-1 promotes cell growth and survival and may delay cellular aging. Importantly, the consequences of aging on cardiac stem cell (CSC) growth and senescence were evaluated. Gene products implicated in growth arrest and senescence, such as p27 Kip1 , p53, p16 INK4a , and p19 ARF , were detected in myocytes of young WT mice, and their expression increased with age. IGF-1 attenuated the levels of these proteins at all ages. Telomerase activity decreased in aging WT myocytes but increased in TG, paralleling the changes in Akt phosphorylation. Reduction in nuclear phospho-Akt and telomerase resulted in telomere shortening and uncapping in WT myocytes. Senescence and death of CSCs increased with age in WT impairing the growth and turnover of cells in the heart. DNA damage and myocyte death exceeded cell formation in old WT, leading to a decreased number of myocytes and heart failure. This did not occur in TG in which CSC-mediated myocyte regeneration compensated for the extent of cell death preventing ventricular dysfunction. IGF-1 enhanced nuclear phospho-Akt and telomerase delaying cellular aging and death. The differential response of TG mice to chronological age may result from preservation of functional CSCs undergoing myocyte commitment. In conclusion, senescence of CSCs and myocytes conditions the development of an aging myopathy. Key Words: telomerase Ⅲ telomere dysfunction Ⅲ cellular senescence T he accepted but never proven paradigm is that the heart is a postmitotic organ characterized by a predetermined number of myocytes, which is defined shortly after birth and is preserved throughout life till death of the organism. 1 According to this view, age of cardiomyocytes corresponds to the age of the organ and organism, ie, without exception, cellular, organ, and organism age coincide. Myocytes must age at the same pace and, at any given time, the heart should be composed of a homogeneous population of myocytes of identical age. Therefore, myocardial aging has been interpreted as a time-dependent biological process that interacts with ischemic heart disease, hypertension, diabetes, and other pathological conditions, which together define the clinical phenotype. 2 The possibility that cardiac aging is an independent determinant of morbidity and mortality has faced opposition and emphasis has been placed on age-associated changes, which increase the chances of cardiovascular events in the elderly. Treatment of cardiac diseases in old patients has resulted in a prolongation of average lifespan. However, maximum lifespan has not increased in the last 70 years, 3 suggesting that cellular aging may play a more important role than generally expected.Several...

In this study, we tested whether the human heart possesses a cardiac stem cell (CSC) pool that promotes regeneration after infarction. For this purpose, CSC growth and senescence were measured in 20 hearts with acute infarcts, 20 hearts with end-stage postinfarction cardiomyopathy, and 12 control hearts. CSC number increased markedly in acute and, to a lesser extent, in chronic infarcts. CSC growth correlated with the increase in telomerasecompetent dividing CSCs from 1.5% in controls to 28% in acute infarcts and 14% in chronic infarcts. The CSC mitotic index increased 29-fold in acute and 14-fold in chronic infarcts. CSCs committed to the myocyte, smooth muscle, and endothelial cell lineages increased Ϸ85-fold in acute infarcts and Ϸ25-fold in chronic infarcts. However, p16 INK4a -p53-positive senescent CSCs also increased and were 10%, 18%, and 40% in controls, acute infarcts, and chronic infarcts, respectively. Old CSCs had short telomeres and apoptosis involved 0.3%, 3.8%, and 9.6% of CSCs in controls, acute infarcts, and chronic infarcts, respectively. These variables reduced the number of functionally competent CSCs from Ϸ26,000͞cm 3 of viable myocardium in acute to Ϸ7,000͞cm 3 in chronic infarcts, respectively. In seven acute infarcts, foci of spontaneous myocardial regeneration that did not involve cell fusion were identified. In conclusion, the human heart possesses a CSC compartment, and CSC activation occurs in response to ischemic injury. The loss of functionally competent CSCs in chronic ischemic cardiomyopathy may underlie the progressive functional deterioration and the onset of terminal failure.cardiac progenitor cells ͉ human heart ͉ myocardial infarction M yocardial regeneration occurs in humans after ischemic injury (1, 2), and myocyte proliferation appears to be restricted to the viable myocardium adjacent to and remote from the infarct (2). The identification of cardiac stem cells (CSCs) in the adult heart (3-7) suggests that replicating myocytes may constitute a subpopulation of rapidly growing amplifying cells originated from more primitive cells. CSCs are distributed throughout the heart, raising the possibility that those located within the infarct or in its proximity could divide and differentiate reconstituting dead myocardium. If this hypothesis were the case, strategies may be developed to enhance myocardial growth promoting partial restoration of the infarct. This response would reduce infarct size, improve function, and decrease mortality. Myocardial regeneration within the infarct could have escaped earlier observations because the heart was not viewed as a self-renewing organ, and myocyte replacement was considered to be regulated by a subset of cells capable of a few rounds of doubling, located by necessity in the spared portion of the ventricle (2). Alternatively, the lack of myocardial regeneration might reflect CSC death within the infarct and͞or the inability of CSCs to migrate and reach the necrotic area. Thus far, no evidence has been presented that CSCs can reconstitute i...

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)-cMet 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 ␣-sarcomeric actin, cardiac myosin heavy chain, troponin I, and ␣-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.cardiac stem cells ͉ myocardial infarction ͉ myocardial regeneration

Abstract-Cardiac stem cells and early committed cells (CSCs-ECCs) express c-Met and insulin-like growth factor-1 (IGF-1) receptors and synthesize and secrete the corresponding ligands, hepatocyte growth factor (HGF) and IGF-1. HGF mobilizes CSCs-ECCs and IGF-1 promotes their survival and proliferation. Therefore, HGF and IGF-1 were injected in the hearts of infarcted mice to favor, respectively, the translocation of CSCs-ECCs from the surrounding myocardium to the dead tissue and the viability and growth of these cells within the damaged area. To facilitate migration and homing of CSCs-ECCs to the infarct, a growth factor gradient was introduced between the site of storage of primitive cells in the atria and the region bordering the infarct. The newly-formed myocardium contained arterioles, capillaries, and functionally competent myocytes that with time increased in size, improving ventricular performance at healing and long thereafter. The volume of regenerated myocytes was 2200 m 3 at 16 days after treatment and reached 5100 m 3 at 4 months. In this interval, nearly 20% of myocytes reached the adult phenotype, varying in size from 10 000 to 20 000 m 3 . Moreover, there were 43Ϯ13 arterioles and 155Ϯ48 capillaries/mm 2 myocardium at 16 days, and 31Ϯ6 arterioles and 390Ϯ56 capillaries at 4 months. Myocardial regeneration induced increased survival and rescued animals with infarcts that were up to 86% of the ventricle, which are commonly fatal. In conclusion, the heart has an endogenous reserve of CSCs-ECCs that can be activated to reconstitute dead myocardium and recover cardiac function. (Circ Res. 2005;97:663-673.)Key Words: cardiac progenitor cells Ⅲ myocardial regeneration Ⅲ mortality A dult cardiac stem cells (CSCs) and early committed cells (ECCs) express the stem cell antigens c-kit, MDR1, and Sca-1. 1-4 c-kit POS cells are self-renewing, clonogenic, and multipotent and give rise to myocytes, smooth muscle cells (SMCs), and endothelial cells (ECs) in vitro and in vivo. 1,5 A similar category of CSCs-ECCs has been found in the human heart, 6,7 suggesting that these undifferentiated cells participate in the normal turnover of cardiac cells and, under favorable conditions, have the ability to form myocytes, coronary arterioles, and capillary structures. 1,5,7 The presence of CSCs-ECCs raises the question of why they fail to respond to ischemic injury with regeneration of myocytes and coronary vessels and restoration of function. CSCs-ECCs distributed within the damaged area may die together with parenchymal cells, however, and SMCs and ECs in coronary vessels prevent myocardial repair. For this reason, we have explored the possibility that CSCs-ECCs, if properly activated, can translocate to sites of damage, survive the unfavorable environment, multiply, and differentiate, forming functionally competent myocardium.Hepatocyte growth factor (HGF) stimulates cell migration 8 by expression of metalloproteinases (MMPs) 9 that by breaking down the extracellular matrix favor cell locomotion, homing, and tissue reco...

Abstract-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. Key Words: transdifferentiation Ⅲ myocardial regeneration Ⅲ cell fusion S everal studies have suggested that adult bone marrow cells (BMCs) can differentiate into cell lineages distinct from the organ in which they reside. 1 The recognition that BMCs maintain some of the growth potential of younger cells has promoted a heated debate about stem cell plasticity and the utilization of BMCs in the treatment of ischemic heart failure. 2 The efficacy of BMCs for myocardial regeneration after infarction was documented 3 years ago, 3 and this protocol was rapidly applied clinically. 4 Nine clinical trials have been completed and several are ongoing and, with the exception of one, 5 all other show positive results. 4,6 -12 Because of the difficulty to demonstrate myocardial regeneration in humans in the absence of cardiac biopsies, three possibilities have been raised in the interpretation of the improvement of cardiac function in patients. They include the development of coronary vessels that rescue hibernating myocardium, 11,12 de novo formation of myocytes 8,10 and vascular structures 4,8,9,12 or the activation and growth of resident progenitor cells via a paracrine effect 12 mediated by BMCs. These are important biological and clinical questions that can be addressed experimentally to acquire a better understanding of the re...

Abstract-Heart failure is the leading cause of death in the elderly, but whether this is the result of a primary aging myopathy dictated by depletion of the cardiac progenitor cell (CPC) pool is unknown. Similarly, whether current lifespan reflects the ineluctable genetic clock or heart failure interferes with the genetically determined fate of the organ and organism is an important question. We have identified that chronological age leads to telomeric shortening in CPCs, which by necessity generate a differentiated progeny that rapidly acquires the senescent phenotype conditioning organ aging. CPC aging is mediated by attenuation of the insulin-like growth factor-1/insulin-like growth factor-1 receptor and hepatocyte growth factor/c-Met systems, which do not counteract any longer the CPC renin-angiotensin system, resulting in cellular senescence, growth arrest, and apoptosis. However, pulse-chase 5-bromodeoxyuridine-labeling assay revealed that the senescent heart contains functionally competent CPCs that have the properties of stem cells. This subset of telomerasecompetent CPCs have long telomeres and, following activation, migrate to the regions of damage, where they generate a population of young cardiomyocytes, reversing partly the aging myopathy. The senescent heart phenotype and heart failure are corrected to some extent, leading to prolongation of maximum lifespan. (Circ Res. 2008;102:597-606.)

Abstract-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. Key Words: Akt Ⅲ apoptosis Ⅲ nuclear Ⅲ cardiomyocytes Ⅲ transgenic P rogrammed cell death, also known as apoptosis, occurs in a wide variety of cardiovascular disorders and is now recognized as a fundamental process that contributes to deterioration of cardiac function. 1 Controlling myocardial cell loss by enhancing survival signal cascades leading to inhibition of apoptosis could be a useful strategy for slowing development of heart failure. Among numerous signaling pathways involved in regulation of cell survival cascades, the serine-threonine kinase Akt/PKB plays a crucial role. 2,3 Cytosolic Akt is activated by phosphorylation mediated by PI3-kinase and 3-phosphoinositide-dependent kinase (PDK) that are stimulated by growth factors such as IGF-1 at cell membrane. 4 -7 After activation, Akt accumulates in the nucleus, phosphorylates multiple protein substrates, and is thought to regulate gene transcription. 8 -19 Akt activation also promotes glucose transport, 20 glycogen 21 and protein 22,23 synthesis, and withdrawal from the cell cycle. 24,25 However, cardiovascular-related Akt research has been predominantly fueled by the ability of activated Akt to enhance cell survival by promoting signaling cascades that lead to inhibition of cardiomyo...

Smooth muscle cell apoptosis with bicuspid aortic valve stenosis occurred before overt aortic dilation, mainly at the convexity, where wall stress is expectedly higher. In this setting, a matrix-dependent proapoptotic signaling was evidenced by increased Bcl-2-modifying factor-Bcl-2 binding. Stress-dependent bicuspid aortic valve matrix changes may trigger early apoptosis by inducing cytoskeletal rearrangement.