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Notch receptor signaling is active during cardiac development and silenced in myocytes after birth. Conversely, outward K + Kv currents progressively appear in postnatal myocytes leading to shortening of the action potential (AP) and acquisition of the mature electrical phenotype. In the present study, we tested the possibility that Notch signaling modulates the electrical behavior of cardiomyocytes by interfering with Kv currents. For this purpose, the effects of Notch receptor activity on electrophysiological properties of myocytes were evaluated using transgenic mice with inducible expression of the Notch1 intracellular domain (NICD), the functional fragment of the activated Notch receptor, and in neonatal myocytes after inhibition of the Notch transduction pathway. By patch clamp, NICD-overexpressing cells presented prolonged AP duration and reduced upstroke amplitude, properties that were coupled with reduced rapidly activating Kv and fast Na + currents, compared with cells obtained from wild-type mice. In cultured neonatal myocytes, inhibition of the proteolitic release of NICD with a γ-secretase antagonist increased transcript levels of the Kv channel-interacting proteins 2 (KChIP2) and enhanced the density of Kv currents. Collectively, these results indicate that Notch signaling represents an important regulator of the electrophysiological behavior of developing and adult myocytes by repressing, at least in part, repolarizing Kv currents. NEW & NOTEWORTHY We investigated the effects of Notch receptor signaling on the electrical properties of cardiomyocytes. Our results indicate that the Notch transduction pathway interferes with outward K + Kv currents, critical determinants of the electrical repolarization of myocytes.

The plasticity of c-kit-positive bone marrow cells (c-kit-BMCs) in tissues different from their organ of origin remains unclear. We tested the hypothesis that c-kit-BMCs are functionally heterogeneous and only a subgroup of these cells possesses cardiomyogenic potential. Population-based assays fall short of identifying the properties of individual stem cells, imposing on us the introduction of single cell-based approaches to track the fate of c-kit-BMCs in the injured heart; they included viral gene-tagging, multicolor clonal-marking and transcriptional profiling. Based on these strategies, we report that single mouse c-kit-BMCs expand clonally within the infarcted myocardium and differentiate into specialized cardiac cells. Newly-formed cardiomyocytes, endothelial cells, fibroblasts and c-kit-BMCs showed in their genome common sites of viral integration, providing strong evidence in favor of the plasticity of a subset of BMCs expressing the c-kit receptor. Similarly, individual c-kit-BMCs, which were infected with multicolor reporters and injected in infarcted hearts, formed cardiomyocytes and vascular cells organized in clusters of similarly colored cells. The uniform distribution of fluorescent proteins in groups of specialized cells documented the polyclonal nature of myocardial regeneration. The transcriptional profile of myogenic c-kit-BMCs and whole c-kit-BMCs was defined by RNA sequencing. Genes relevant for engraftment, survival, migration, and differentiation were enriched in myogenic c-kit-BMCs, a cell subtype which could not be assigned to a specific hematopoietic lineage. Collectively, our findings demonstrate that the bone marrow comprises a category of cardiomyogenic, vasculogenic and/or fibrogenic c-kit-positive cells and a category of c-kit-positive cells that retains an undifferentiated state within the damaged heart., Competing Interests: P.A. is a member of Autologous Regeneration LLP. P.A. and A.L. are members of AAL Scientifics Inc. The remaining authors declare no competing financial interests.

p53 is an important modulator of stem cell fate, but its role in cardiac progenitor cells (CPCs) is unknown. Here, we tested the effects of a single extra-copy of p53 on the function of CPCs in the presence of oxidative stress mediated by doxorubicin in vitro and type-1 diabetes in vivo. CPCs were obtained from super-p53 transgenic mice (p53-tg), in which the additional allele is regulated in a manner similar to the endogenous protein. Old CPCs with increased p53 dosage showed a superior ability to sustain oxidative stress, repair DNA damage and restore cell division. With doxorubicin, a larger fraction of CPCs carrying an extra-copy of the p53 allele recruited γH2A.X reestablishing DNA integrity. Enhanced p53 expression resulted in a superior tolerance to oxidative stress in vivo by providing CPCs with defense mechanisms necessary to survive in the milieu of the diabetic heart; they engrafted in regions of tissue injury and in three days acquired the cardiomyocyte phenotype. The biological advantage provided by the increased dosage of p53 in CPCs suggests that this genetic strategy may be translated to humans to increase cellular engraftment and growth, critical determinants of successful cell therapy for the failing heart., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Diabetes and other metabolic conditions characterized by elevated blood glucose constitute important risk factors for cardiovascular disease. Hyperglycemia targets myocardial cells rendering ineffective mechanical properties of the heart, but cellular alterations dictating the progressive deterioration of cardiac function with metabolic disorders remain to be clarified. In the current study, we examined the effects of hyperglycemia on cardiac function and myocyte physiology by employing mice with high blood glucose induced by administration of streptozotocin, a compound toxic to insulin-producing β-cells. We found that hyperglycemia initially delayed the electrical recovery of the heart, whereas cardiac function became defective only after ~2 mo with this condition and gradually worsened with time. Prolonged hyperglycemia was associated with increased chamber dilation, thinning of the left ventricle (LV), and myocyte loss. Cardiomyocytes from hyperglycemic mice exhibited defective Ca 2+ transients before the appearance of LV systolic defects. Alterations in Ca 2+ transients involved enhanced spontaneous Ca 2+ releases from the sarcoplasmic reticulum (SR), reduced cytoplasmic Ca 2+ clearance, and declined SR Ca 2+ load. These defects have important consequences on myocyte contraction, relaxation, and mechanisms of rate adaptation. Collectively, our data indicate that hyperglycemia alters intracellular Ca 2+ homeostasis in cardiomyocytes, hindering contractile activity and contributing to the manifestation of the diabetic cardiomyopathy., New & Noteworthy: We have investigated the effects of hyperglycemia on cardiomyocyte physiology and ventricular function. Our results indicate that defective Ca 2+ handling is a critical component of the progressive deterioration of cardiac performance of the diabetic heart., (Copyright © 2017 the American Physiological Society.)

Studies of myocardial aging are complex and the mechanisms involved in the deterioration of ventricular performance and decreased functional reserve of the old heart remain to be properly defined. We have studied a colony of beagle dogs from 3 to 14 yr of age kept under a highly regulated environment to define the effects of aging on the myocardium. Ventricular, myocardial, and myocyte function, together with anatomical and structural properties of the organ and cardiomyocytes, were evaluated. Ventricular hypertrophy was not observed with aging and the structural composition of the myocardium was modestly affected. Alterations in the myocyte compartment were identified in aged dogs, and these factors negatively interfere with the contractile reserve typical of the young heart. The duration of the action potential is prolonged in old cardiomyocytes contributing to the slower electrical recovery of the myocardium. Also, the remodeled repolarization of cardiomyocytes with aging provides inotropic support to the senescent muscle but compromises its contractile reserve, rendering the old heart ineffective under conditions of high hemodynamic demand. The defects in the electrical and mechanical properties of cardiomyocytes with aging suggest that this cell population is an important determinant of the cardiac senescent phenotype. Collectively, the delayed electrical repolarization of aging cardiomyocytes may be viewed as a critical variable of the aging myopathy and its propensity to evolve into ventricular decompensation under stressful conditions., (Copyright © 2016 the American Physiological Society.)

Background: Diabetes is associated with prolongation of the QT interval of the electrocardiogram and enhanced dispersion of ventricular repolarization, factors that, together with atherosclerosis and myocardial ischemia, may promote the occurrence of electrical disorders. Thus, we tested the possibility that alterations in transmembrane ionic currents reduce the repolarization reserve of myocytes, leading to action potential (AP) prolongation and enhanced beat-to-beat variability of repolarization., Methods and Results: Diabetes was induced in mice with streptozotocin (STZ), and effects of hyperglycemia on electrical properties of whole heart and myocytes were studied with respect to an untreated control group (Ctrl) using electrocardiographic recordings in vivo, ex vivo perfused hearts, and single-cell patch-clamp analysis. Additionally, a newly developed algorithm was introduced to obtain detailed information of the impact of high glucose on AP profile. Compared to Ctrl, hyperglycemia in STZ-treated animals was coupled with prolongation of the QT interval, enhanced temporal dispersion of electrical recovery, and susceptibility to ventricular arrhythmias, defects observed, in part, in the Akita mutant mouse model of type I diabetes. AP was prolonged and beat-to-beat variability of repolarization was enhanced in diabetic myocytes, with respect to Ctrl cells. Density of Kv K(+) and L-type Ca(2+) currents were decreased in STZ myocytes, in comparison to cells from normoglycemic mice. Pharmacological reduction of Kv currents in Ctrl cells lengthened AP duration and increased temporal dispersion of repolarization, reiterating features identified in diabetic myocytes., Conclusions: Reductions in the repolarizing K(+) currents may contribute to electrical disturbances of the diabetic heart., (© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Striated preferentially expressed gene (Speg) is a member of the myosin light chain kinase family. We previously showed that disruption of the Speg gene locus in mice leads to a dilated cardiomyopathy with immature-appearing cardiomyocytes. Here we show that cardiomyopathy of Speg(-/-) mice arises as a consequence of defects in cardiac progenitor cell (CPC) function, and that neonatal cardiac dysfunction can be rescued by in utero injections of wild-type CPCs into Speg(-/-) foetal hearts. CPCs harvested from Speg(-/-) mice display defects in clone formation, growth and differentiation into cardiomyocytes in vitro, which are associated with cardiac dysfunction in vivo. In utero administration of wild-type CPCs into the hearts of Speg(-/-) mice results in CPC engraftment, differentiation and myocardial maturation, which rescues Speg(-/-) mice from neonatal heart failure and increases the number of live births by fivefold. We propose that in utero administration of CPCs may have future implications for treatment of neonatal heart diseases.

The aging myopathy manifests itself with diastolic dysfunction and preserved ejection fraction. We raised the possibility that, in a mouse model of physiological aging, defects in electromechanical properties of cardiomyocytes are important determinants of the diastolic characteristics of the myocardium, independently from changes in structural composition of the muscle and collagen framework. Here we show that an increase in the late Na(+) current (INaL) in aging cardiomyocytes prolongs the action potential (AP) and influences temporal kinetics of Ca(2+) cycling and contractility. These alterations increase force development and passive tension. Inhibition of INaL shortens the AP and corrects dynamics of Ca(2+) transient, cell contraction and relaxation. Similarly, repolarization and diastolic tension of the senescent myocardium are partly restored. Thus, INaL offers inotropic support, but negatively interferes with cellular and ventricular compliance, providing a new perspective of the biology of myocardial aging and the aetiology of the defective cardiac performance in the elderly.

Cardiac aging has been confounded by the concept that the heart is a postmitotic organ characterized by a predetermined number of myocytes, which is established at birth and largely preserved throughout life until death of the organ and organism. Based on this premise, the age of cardiac cells should coincide with that of the organism; at any given time, the heart would be composed of a homogeneous population of myocytes of identical age. The discovery that stem cells reside in the heart and generate cardiac cell lineages has imposed a reconsideration of the mechanisms implicated in the manifestations of the aging myopathy. The progressive alterations of terminally differentiated myocytes, and vascular smooth muscle cells and endothelial cells may represent an epiphenomenon dictated by aging effects on cardiac progenitor cells (CPCs). Changes in the properties of CPCs with time may involve loss of self-renewing capacity, increased symmetric division with formation of daughter committed cells, partial depletion of the primitive pool, biased differentiation to the fibroblast fate, impaired ability to migrate, and forced entry into an irreversible quiescent state. Telomere shortening is a major variable of cellular senescence and organ aging, and support the notion that CPCs with critically shortened or dysfunctional telomeres contribute to myocardial aging and chronic heart failure. These defects constitute the critical variables that define the aging myopathy in humans. Importantly, a compartment of functionally competent human CPCs persists in the decompensated heart pointing to stem cell therapy as a novel form of treatment for the aging myopathy., (Copyright © 2015 John Wiley & Sons, Inc.)

Following the recognition that hematopoietic stem cells improve the outcome of myocardial infarction in animal models, bone marrow mononuclear cells, CD34-positive cells, and mesenchymal stromal cells have been introduced clinically. The intracoronary or intramyocardial injection of these cell classes has been shown to be safe and to produce a modest but significant enhancement in systolic function. However, the identification of resident cardiac stem cells in the human heart (hCSCs) has created great expectation concerning the potential implementation of this category of autologous cells for the management of the human disease. Although phase 1 clinical trials have been conducted with encouraging results, the search for the most powerful hCSC for myocardial regeneration is in its infancy. This manuscript discusses the efforts performed in our laboratory to characterize the critical biological variables that define the growth reserve of hCSCs. Based on the theory of the immortal DNA template, we propose that stem cells retaining the old DNA represent 1 of the most powerful cells for myocardial regeneration. Similarly, the expression of insulin-like growth factor-1 receptors in hCSCs recognizes a cell phenotype with superior replicating reserve. However, the impressive recovery in ventricular hemodynamics and anatomy mediated by clonal hCSCs carrying the "mother" DNA underscores the clinical relevance of this hCSC class for the treatment of human heart failure.

This review article discusses the mechanisms of cardiomyogenesis in the adult heart. They include the re-entry of cardiomyocytes into the cell cycle; dedifferentiation of pre-existing cardiomyocytes, which assume an immature replicating cell phenotype; transdifferentiation of hematopoietic stem cells into cardiomyocytes; and cardiomyocytes derived from activation and lineage specification of resident cardiac stem cells. The recognition of the origin of cardiomyocytes is of critical importance for the development of strategies capable of enhancing the growth response of the myocardium; in fact, cell therapy for the decompensated heart has to be based on the acquisition of this fundamental biological knowledge., (© 2015 American Heart Association, Inc.)

Significance: Heart disease is the primary cause of death in the industrialized world. Cardiac failure is dictated by an uncompensated reduction in the number of viable and fully functional cardiomyocytes. While current pharmacological therapies alleviate the symptoms associated with cardiac deterioration, heart transplantation remains the only therapy for advanced heart failure. Therefore, there is a pressing need for novel therapeutic modalities. Cell-based therapies involving cardiac stem cells (CSCs) constitute a promising emerging approach for the replenishment of the lost tissue and the restoration of cardiac contractility., Recent Advances: CSCs reside in the adult heart and govern myocardial homeostasis and repair after injury by producing new cardiomyocytes and vascular structures. In the last decade, different classes of immature cells expressing distinct stem cell markers have been identified and characterized in terms of their growth properties, differentiation potential, and regenerative ability. Phase I clinical trials, employing autologous CSCs in patients with ischemic cardiomyopathy, are being completed with encouraging results., Critical Issues: Accumulating evidence concerning the role of CSCs in heart regeneration imposes a reconsideration of the mechanisms of cardiac aging and the etiology of heart failure. Deciphering the molecular pathways that prevent activation of CSCs in their environment and understanding the processes that affect CSC survival and regenerative function with cardiac pathologies, commonly accompanied by alterations in redox conditions, are of great clinical importance., Future Directions: Further investigations of CSC biology may be translated into highly effective and novel therapeutic strategies aiming at the enhancement of the endogenous healing capacity of the diseased heart.

The critical role that stem cell niches have in cardiac homeostasis and myocardial repair following injury is the focus of this review. Cardiac niches represent specialized microdomains where the quiescent and activated state of resident stem cells is regulated. Alterations in niche function with aging and cardiac diseases result in abnormal sites of cardiomyogenesis and inadequate myocyte formation. The relevance of Notch1 signaling, gap-junction formation, HIF-1α and metabolic state in the regulation of stem cell growth and differentiation within the cardiac niches are discussed., (Copyright © 2014. Published by Elsevier B.V.)

Fabry disease (angiokeratoma corporis diffusum universale) is a rare, progressive, X-linked lysosomal storage disease. Deficiency of the α-galactosidase A (α-gal A) enzyme leads to accumulation of neutral glycosphingolipids within vascular endothelial lysosomes of various organs, including skin, kidneys, heart, and brain (1). We herein describe the case of a 30-year-old female presenting two classic signs of Fabry disease, angiokeratomas and episodic acroparesthesias, in the absence of other clinical manifestations. An haplotype corresponding to the combination of three different nucleotide polymorphic variants (g. 7192-7198del5+ g. 10115A>G + g. 10956 C>T) at the heterozygous state, was identified (2).

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 with aging and the formation of scarred tissue following myocardial infarction have been interpreted as irrefutable proofs of the post-mitotic characteristics of the adult heart. However, emerging evidence supports a more dynamic view of the myocardium in which cell death and cell restoration are vital components of the remodeling process that governs organ homeostasis, aging and disease. The identification of dividing myocytes throughout the life span of the organisms and the recognition that undifferentiated primitive cells regulate myocyte turnover and tissue regeneration indicate that the heart is a self-renewing organ controlled by a compartment of resident stem cells. Moreover, exogenous progenitors of bone marrow origin transdifferentiate and acquire the cardiomyocyte and vascular lineages. This new reality constitutes the foundation of the numerous cell-based clinical trials that have been conducted in the last decade for the treatment of ischemic and non-ischemic cardiomyopathies., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: The efficacy of bypass surgery in patients with ischemic cardiomyopathy is not easily predictable; preoperative clinical conditions may be similar, but the outcome may differ significantly. We hypothesized that the growth reserve of cardiac stem cells (CSCs) and circulating cytokines promoting CSC activation are critical determinants of ventricular remodeling in this patient population., Methods and Results: To document the growth kinetics of CSCs, population-doubling time, telomere length, telomerase activity, and insulin-like growth factor-1 receptor expression were measured in CSCs isolated from 38 patients undergoing bypass surgery. Additionally, the blood levels of insulin-like growth factor-1, hepatocyte growth factor, and vascular endothelial growth factor were evaluated. The variables of CSC growth were expressed as a function of the changes in wall thickness, chamber diameter and volume, ventricular mass-to-chamber volume ratio, and ejection fraction, before and 12 months after surgery. A high correlation was found between indices of CSC function and cardiac anatomy. Negative ventricular remodeling was not observed if CSCs retained a significant growth reserve. The high concentration of insulin-like growth factor-1 systemically pointed to the insulin-like growth factor-1-insulin-like growth factor-1 receptor system as a major player in the adaptive response of the myocardium. hepatocyte growth factor, a mediator of CSC migration, was also high in these patients preoperatively, as was vascular endothelial growth factor, possibly reflecting the vascular growth needed before bypass surgery. Conversely, a decline in CSC growth was coupled with wall thinning, chamber dilation, and depressed ejection fraction., Conclusions: The telomere-telomerase axis, population-doubling time, and insulin-like growth factor-1 receptor expression in CSCs, together with a high circulating level of insulin-like growth factor-1, represent a novel biomarker able to predict the evolution of ischemic cardiomyopathy following revascularization.

Rationale: Hypoxia favors stem cell quiescence, whereas normoxia is required for stem cell activation, but whether cardiac stem cell (CSC) function is regulated by the hypoxic/normoxic state of the cell is currently unknown., Objective: A balance between hypoxic and normoxic CSCs may be present in the young heart, although this homeostatic control may be disrupted with aging. Defects in tissue oxygenation occur in the old myocardium, and this phenomenon may expand the pool of hypoxic CSCs, which are no longer involved in myocyte renewal., Methods and Results: Here, we show that the senescent heart is characterized by an increased number of quiescent CSCs with intact telomeres that cannot re-enter the cell cycle and form a differentiated progeny. Conversely, myocyte replacement is controlled only by frequently dividing CSCs with shortened telomeres; these CSCs generate a myocyte population that is chronologically young but phenotypically old. Telomere dysfunction dictates their actual age and mechanical behavior. However, the residual subset of quiescent young CSCs can be stimulated in situ by stem cell factor reversing the aging myopathy., Conclusions: Our findings support the notion that strategies targeting CSC activation and growth interfere with the manifestations of myocardial aging in an animal model. Although caution has to be exercised in the translation of animal studies to human beings, our data strongly suggest that a pool of functionally competent CSCs persists in the senescent heart and that this stem cell compartment can promote myocyte regeneration effectively, partly correcting the aging myopathy.

Background: Aging negatively impacts on the function of resident human cardiac progenitor cells (hCPCs). Effective regeneration of the injured heart requires mobilization of hCPCs to the sites of damage. In the young heart, signaling by the guidance receptor EphA2 in response to the ephrin A1 ligand promotes hCPC motility and improves cardiac recovery after infarction., Methods and Results: We report that old hCPCs are characterized by cell-autonomous inhibition of their migratory ability ex vivo and impaired translocation in vivo in the damaged heart. EphA2 expression was not decreased in old hCPCs; however, the elevated level of reactive oxygen species in aged cells induced post-translational modifications of the EphA2 protein. EphA2 oxidation interfered with ephrin A1-stimulated receptor auto-phosphorylation, activation of Src family kinases, and caveolin-1-mediated internalization of the receptor. Cellular aging altered the EphA2 endocytic route, affecting the maturation of EphA2-containing endosomes and causing premature signal termination. Overexpression of functionally intact EphA2 in old hCPCs corrected the defects in endocytosis and downstream signaling, enhancing cell motility. Based on the ability of phenotypically young hCPCs to respond efficiently to ephrin A1, we developed a novel methodology for the prospective isolation of live hCPCs with preserved migratory capacity and growth reserve., Conclusions: Our data demonstrate that the ephrin A1/EphA2 pathway may serve as a target to facilitate trafficking of hCPCs in the senescent myocardium. Importantly, EphA2 receptor function can be implemented for the selection of hCPCs with high therapeutic potential, a clinically relevant strategy that does not require genetic manipulation of stem cells.

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 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., (Copyright © 2013 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.)

Background: Relevant preclinical models are necessary for further mechanistic and translational studies of c-kit+ cardiac stem cells (CSCs). The present study was undertaken to determine whether intracoronary CSCs are beneficial in a porcine model of chronic ischemic cardiomyopathy., Methods and Results: Pigs underwent a 90-minute coronary occlusion followed by reperfusion. Three months later, autologous CSCs (n=11) or vehicle (n=10) were infused into the infarct-related artery. At this time, all indices of left ventricular (LV) function were similar in control and CSC-treated pigs, indicating that the damage inflicted by the infarct in the 2 groups was similar; 1 month later, however, CSC-treated pigs exhibited significantly greater LV ejection fraction (echocardiography) (51.7±2.0% versus 42.9±2.3%, P<0.01), systolic thickening fraction in the infarcted LV wall, and maximum LV dP/dt, as well as lower LV end-diastolic pressure. Confocal microscopy showed clusters of small α-sarcomeric actin-positive cells expressing Ki67 in the scar of treated pigs, consistent with cardiac regeneration. The origin of these cycling myocytes from the injected cells was confirmed in 4 pigs that received enhanced green fluorescent protein -labeled CSCs, which were positive for the cardiac markers troponin I, troponin T, myosin heavy chain, and connexin-43. Some engrafted CSCs also formed vascular structures and expressed α-smooth muscle actin., Conclusions: Intracoronary infusion of autologous CSCs improves regional and global LV function and promotes cardiac and vascular regeneration in pigs with old myocardial infarction (scar). The results mimic those recently reported in humans (Stem Cell Infusion in Patients with Ischemic CardiOmyopathy [SCIPIO] trial) and establish this porcine model of ischemic cardiomyopathy as a useful and clinically relevant model for studying CSCs.

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: Multiple progenitors derived from the heart and bone marrow (BM) have been used for cardiac repair. Despite this, not much is known about the molecular identity and relationship among these progenitors. To develop a robust stem cell therapy for the heart, it is critical to understand the molecular identity of the multiple cardiogenic progenitor cells., Objective: This study is the first report of high-throughput transcriptional profiling of cardiogenic progenitor cells carried out on an identical platform., Method and Results: Microarray-based transcriptional profiling was carried out for 3 cardiac (ckit(+), Sca1(+), and side population) and 2 BM (ckit(+) and mesenchymal stem cell) progenitors, obtained from age- and sex-matched wild-type C57BL/6 mice. Analysis indicated that cardiac-derived ckit(+) population was very distinct from Sca1(+) and side population cells in the downregulation of genes encoding for cell-cell and cell-matrix adhesion proteins, and in the upregulation of developmental genes. Significant enrichment of transcripts involved in DNA replication and repair was observed in BM-derived progenitors. The BM ckit(+) cells seemed to have the least correlation with the other progenitors, with enrichment of immature neutrophil-specific molecules., Conclusions: Our study indicates that cardiac ckit(+) cells represent the most primitive population in the rodent heart. Primitive cells of cardiac versus BM origin differ significantly with respect to stemness and cardiac lineage-specific genes, and molecules involved in DNA replication and repair. The detailed molecular profile of progenitors reported here will serve as a useful reference to determine the molecular identity of progenitors used in future preclinical and clinical studies.

Background: Regenerative medicine, including cell therapy, is a promising strategy for recovery of the damaged myocardium. C-kit-positive cardiac stem cells (CSCs) have been shown to improve myocardial function after ischemic injury in animal models and in early clinical experience. We used a chronic large animal model of myocardial infarction with substantial reductions in left-ventricular (LV) ejection fraction and adverse remodeling to examine the effect of late autologous CSC intramyocardial injection on long-term cardiac structure and function., Methods and Results: Thoracotomy and ligation of the proximal left anterior descending artery, additional diagonal branches, and atrial biopsy for CSC culture were performed in canines. Baseline cardiac MRI was performed at 6 weeks postinfarct followed by repeat thoracotomy for randomization to intramyocardial injection of CSCs (n=13) or vehicle alone (n=6). At 30 weeks postmyocardial infarction, repeat MRI was performed. Data were analyzed using nonparametric tests (Wilcoxon signed-rank and rank-sum tests). In control animals, LV end-systolic volume and end-diastolic volume increased from 6 to 30 weeks (median and interquartile range, 51.3 mL [43.3-57.4] to 76.1 mL [72.0-82.4]; P=0.03 and 78.5 mL [69.7-86.1] to 99.2 mL [97.1-100.4]; P=0.03). Left-ventricular ejection fraction declined further (35.2% [27.9-38.7] to 26.4% [22.0-31.0]; P=0.12). In the cell-treated animals, this late adverse LV remodeling was attenuated (LV end-systolic volume, 42.6 mL [38.5-50.5] to 56.1 mL [50.3-63.0]; P=0.01 versus control). There was a nonsignificant attenuation in the increase in LV end-diastolic volume (64.8 mL [60.7-71.3] to 83.5 mL [74.7-90.8]; P=0.14 versus control) and LV ejection fraction change over time differed (30.5% [28.4-33.4] to 32.9% [28.6-36.9]; P=0.04 versus control)., Conclusions: Intramyocardial injection of autologous CSCs in a late phase model of chronic infarction resulted in less increase in LV end-systolic volume and preservation of LV ejection fraction.

This article discusses current understanding of myocardial biology, emphasizing the regeneration potential of the adult human heart and the mechanisms involved. In the last decade, a novel conceptual view has emerged. The heart is no longer considered a postmitotic organ, but is viewed as a self-renewing organ characterized by a resident stem cell compartment responsible for tissue homeostasis and cardiac repair following injury. Additionally, HSCs possess the ability to transdifferentiate and acquire the cardiomyocyte, vascular endothelial, and smooth muscle cell lineages. Both cardiac and hematopoietic stem cells may be used therapeutically in an attempt to reverse the devastating consequences of chronic heart failure of ischemic and nonischemic origin.

Background: Two opposite views of cardiac growth are currently held; one views the heart as a static organ characterized by a large number of cardiomyocytes that are present at birth and live as long as the organism, and the other views the heart a highly plastic organ in which the myocyte compartment is restored several times during the course of life., Methods and Results: The average age of cardiomyocytes, vascular endothelial cells (ECs), and fibroblasts and their turnover rates were measured by retrospective (14)C birth dating of cells in 19 normal hearts 2 to 78 years of age and in 17 explanted failing hearts 22 to 70 years of age. We report that the human heart is characterized by a significant turnover of ventricular myocytes, ECs, and fibroblasts, physiologically and pathologically. Myocyte, EC, and fibroblast renewal is very high shortly after birth, decreases during postnatal maturation, remains relatively constant in the adult organ, and increases dramatically with age. From 20 to 78 years of age, the adult human heart entirely replaces its myocyte, EC, and fibroblast compartment ≈8, ≈6, and ≈8 times, respectively. Myocyte, EC, and fibroblast regeneration is further enhanced with chronic heart failure., Conclusions: The human heart is a highly dynamic organ that retains a remarkable degree of plasticity throughout life and in the presence of chronic heart failure. However, the ability to regenerate cardiomyocytes, vascular ECs, and fibroblasts cannot prevent the manifestations of myocardial aging or oppose the negative effects of ischemic and idiopathic dilated cardiomyopathy.

Rationale: According to the immortal DNA strand hypothesis, dividing stem cells selectively segregate chromosomes carrying the old template DNA, opposing accumulation of mutations resulting from nonrepaired replication errors and attenuating telomere shortening., Objective: Based on the premise of the immortal DNA strand hypothesis, we propose that stem cells retaining the old DNA would represent the most powerful cells for myocardial regeneration., Methods and Results: Division of human cardiac stem cells (hCSCs) by nonrandom and random segregation of chromatids was documented by clonal assay of bromodeoxyuridine-tagged hCSCs. Additionally, their growth properties were determined by a series of in vitro and in vivo studies. We report that a small class of hCSCs retain during replication the mother DNA and generate 2 daughter cells, which carry the old and new DNA, respectively. hCSCs with immortal DNA form a pool of nonsenescent cells with longer telomeres and higher proliferative capacity. The self-renewal and long-term repopulating ability of these cells was shown in serial-transplantation assays in the infarcted heart; these cells created a chimeric organ, composed of spared rat and regenerated human cardiomyocytes and coronary vessels, leading to a remarkable restoration of cardiac structure and function. The documentation that hCSCs divide by asymmetrical and symmetrical chromatid segregation supports the view that the human heart is a self-renewing organ regulated by a compartment of resident hCSCs., Conclusions: The impressive recovery in ventricular hemodynamics and anatomy mediated by clonal hCSCs carrying the "mother" DNA underscores the clinical relevance of this stem cell class for the management of heart failure in humans.

Rationale: Embryonic and fetal myocardial growth is characterized by a dramatic increase in myocyte number, but whether the expansion of the myocyte compartment is dictated by activation and commitment of resident cardiac stem cells (CSCs), division of immature myocytes or both is currently unknown., Objective: In this study, we tested whether prenatal cardiac development is controlled by activation and differentiation of CSCs and whether division of c-kit-positive CSCs in the mouse heart is triggered by spontaneous Ca(2+) oscillations., Methods and Results: We report that embryonic-fetal c-kit-positive CSCs are self-renewing, clonogenic and multipotent in vitro and in vivo. The growth and commitment of c-kit-positive CSCs is responsible for the generation of the myocyte progeny of the developing heart. The close correspondence between values computed by mathematical modeling and direct measurements of myocyte number at E9, E14, E19 and 1 day after birth strongly suggests that the organogenesis of the embryonic heart is dependent on a hierarchical model of cell differentiation regulated by resident CSCs. The growth promoting effects of c-kit-positive CSCs are triggered by spontaneous oscillations in intracellular Ca(2+), mediated by IP3 receptor activation, which condition asymmetrical stem cell division and myocyte lineage specification., Conclusions: Myocyte formation derived from CSC differentiation is the major determinant of cardiac growth during development. Division of c-kit-positive CSCs in the mouse is promoted by spontaneous Ca(2+) spikes, which dictate the pattern of stem cell replication and the generation of a myocyte progeny at all phases of prenatal life and up to one day after birth.

Background: c-kit-positive, lineage-negative cardiac stem cells (CSCs) improve post-infarction left ventricular (LV) dysfunction when administered to animals. We undertook a phase 1 trial (Stem Cell Infusion in Patients with Ischemic cardiOmyopathy [SCIPIO]) of autologous CSCs for the treatment of heart failure resulting from ischaemic heart disease., Methods: In stage A of the SCIPIO trial, patients with post-infarction LV dysfunction (ejection fraction [EF] ≤40%) before coronary artery bypass grafting were consecutively enrolled in the treatment and control groups. In stage B, patients were randomly assigned to the treatment or control group in a 2:3 ratio by use of a computer-generated block randomisation scheme. 1 million autologous CSCs were administered by intracoronary infusion at a mean of 113 days (SE 4) after surgery; controls were not given any treatment. Although the study was open label, the echocardiographic analyses were masked to group assignment. The primary endpoint was short-term safety of CSCs and the secondary endpoint was efficacy. A per-protocol analysis was used. This study is registered with ClinicalTrials.gov, number NCT00474461., Findings: This study is still in progress. 16 patients were assigned to the treatment group and seven to the control group; no CSC-related adverse effects were reported. In 14 CSC-treated patients who were analysed, LVEF increased from 30·3% (SE 1·9) before CSC infusion to 38·5% (2·8) at 4 months after infusion (p=0·001). By contrast, in seven control patients, during the corresponding time interval, LVEF did not change (30·1% [2·4] at 4 months after CABG vs 30·2% [2·5] at 8 months after CABG). Importantly, the salubrious effects of CSCs were even more pronounced at 1 year in eight patients (eg, LVEF increased by 12·3 ejection fraction units [2·1] vs baseline, p=0·0007). In the seven treated patients in whom cardiac MRI could be done, infarct size decreased from 32·6 g (6·3) by 7·8 g (1·7; 24%) at 4 months (p=0·004) and 9·8 g (3·5; 30%) at 1 year (p=0·04)., Interpretation: These initial results in patients are very encouraging. They suggest that intracoronary infusion of autologous CSCs is effective in improving LV systolic function and reducing infarct size in patients with heart failure after myocardial infarction, and warrant further, larger, phase 2 studies., Funding: University of Louisville Research Foundation and National Institutes of Health., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Human ischemic cardiomyopathy is characterized by de novo cardiomyogenesis, which is limited to the surviving portion of the ventricle, and by organ hypertrophy that develops as a chronic response to ischemic injury. Although myocyte hypertrophy and myocyte regeneration restore the original myocardial mass, the coronary vasculature remains defective and the extent and regulation of myocardial perfusion are severely impaired. Recently, vascular stem cells (VSCs) have been identified in the coronary circulation. VSCs express c-kit and the vascular endothelial growth factor receptor-2, KDR. These cells are self-renewing, clonogenic, and multipotent in vitro and in vivo. In animal models of critical coronary artery stenosis, VSCs form large conductive coronary arteries and their distal branches. This degree of vasculogenesis replaces partly the function of the occluded coronary artery improving myocardial perfusion and positively interfering with the development of the post-infarction myopathy. Cell therapy directed to the restoration of the integrity of the coronary circulation, the replacement of atherosclerotic coronary vessels, or both, would change dramatically the goal of cell therapy for the ischemic heart: the prevention of myocardial injury would become the end-point of cell therapy rather than the partial recovery of established damage.

For nearly a century, the human heart has been viewed as a terminally differentiated postmitotic organ in which the number of cardiomyocytes is established at birth, and these cells persist throughout the lifespan of the organ and organism. However, the discovery that cardiac stem cells live in the heart and differentiate into the various cardiac cell lineages has changed profoundly our understanding of myocardial biology. Cardiac stem cells regulate myocyte turnover and condition myocardial recovery after injury. This novel information imposes a reconsideration of the mechanisms involved in myocardial aging and the progression of cardiac hypertrophy to heart failure. Similarly, the processes implicated in the adaptation of the infarcted heart have to be dissected in terms of the critical role that cardiac stem cells and myocyte regeneration play in the restoration of myocardial mass and ventricular function. Several categories of cardiac progenitors have been described but, thus far, the c-kit-positive cell is the only class of resident cells with the biological and functional properties of tissue specific adult stem cells.

Currently, it is unknown whether defects in stem cell growth and differentiation contribute to myocardial aging and chronic heart failure (CHF), and whether a compartment of functional human cardiac stem cells (hCSCs) persists in the decompensated heart. To determine whether aging and CHF are critical determinants of the loss in growth reserve of the heart, the properties of hCSCs were evaluated in 18 control and 23 explanted hearts. Age and CHF showed a progressive decrease in functionally competent hCSCs. Chronological age was a major predictor of five biomarkers of hCSC senescence: telomeric shortening, attenuated telomerase activity, telomere dysfunction-induced foci, and p21(Cip1) and p16(INK4a) expression. CHF had similar consequences for hCSCs, suggesting that defects in the balance between cardiomyocyte mass and the pool of nonsenescent hCSCs may condition the evolution of the decompensated myopathy. A correlation was found previously between telomere length in circulating bone marrow cells and cardiovascular diseases, but that analysis was restricted to average telomere length in a cell population, neglecting the fact that telomere attrition does not occur uniformly in all cells. The present study provides the first demonstration that dysfunctional telomeres in hCSCs are biomarkers of aging and heart failure. The biomarkers of cellular senescence identified here can be used to define the birth date of hCSCs and to sort young cells with potential therapeutic efficacy., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Rationale: Age and coronary artery disease may negatively affect the function of human cardiac stem cells (hCSCs) and their potential therapeutic efficacy for autologous cell transplantation in the failing heart., Objective: Insulin-like growth factor (IGF)-1, IGF-2, and angiotensin II (Ang II), as well as their receptors, IGF-1R, IGF-2R, and AT1R, were characterized in c-kit(+) hCSCs to establish whether these systems would allow us to separate hCSC classes with different growth reserve in the aging and diseased myocardium., Methods and Results: C-kit(+) hCSCs were collected from myocardial samples obtained from 24 patients, 48 to 86 years of age, undergoing elective cardiac surgery for coronary artery disease. The expression of IGF-1R in hCSCs recognized a young cell phenotype defined by long telomeres, high telomerase activity, enhanced cell proliferation, and attenuated apoptosis. In addition to IGF-1, IGF-1R(+) hCSCs secreted IGF-2 that promoted myocyte differentiation. Conversely, the presence of IGF-2R and AT1R, in the absence of IGF-1R, identified senescent hCSCs with impaired growth reserve and increased susceptibility to apoptosis. The ability of IGF-1R(+) hCSCs to regenerate infarcted myocardium was then compared with that of unselected c-kit(+) hCSCs. IGF-1R(+) hCSCs improved cardiomyogenesis and vasculogenesis. Pretreatment of IGF-1R(+) hCSCs with IGF-2 resulted in the formation of more mature myocytes and superior recovery of ventricular structure., Conclusions: hCSCs expressing only IGF-1R synthesize both IGF-1 and IGF-2, which are potent modulators of stem cell replication, commitment to the myocyte lineage, and myocyte differentiation, which points to this hCSC subset as the ideal candidate cell for the management of human heart failure.

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: Understanding the mechanisms that regulate trafficking of human cardiac stem cells (hCSCs) may lead to development of new therapeutic approaches for the failing heart., Objective: We tested whether the motility of hCSCs in immunosuppressed infarcted animals is controlled by the guidance system that involves the interaction of Eph receptors with ephrin ligands., Methods and Results: Within the cardiac niches, cardiomyocytes expressed preferentially the ephrin A1 ligand, whereas hCSCs possessed the EphA2 receptor. Treatment of hCSCs with ephrin A1 resulted in the rapid internalization of the ephrin A1-EphA2 complex, posttranslational modifications of Src kinases, and morphological changes consistent with the acquisition of a motile cell phenotype. Ephrin A1 enhanced the motility of hCSCs in vitro, and their migration in vivo following acute myocardial infarction. At 2 weeks after infarction, the volume of the regenerated myocardium was 2-fold larger in animals injected with ephrin A1-activated hCSCs than in animals receiving control hCSCs; this difference was dictated by a greater number of newly formed cardiomyocytes and coronary vessels. The increased recovery in myocardial mass with ephrin A1-treated hCSCs was characterized by further restoration of cardiac function and by a reduction in arrhythmic events., Conclusions: Ephrin A1 promotes the motility of EphA2-positive hCSCs, facilitates their migration to the area of damage, and enhances cardiac repair. Thus, in situ stimulation of resident hCSCs with ephrin A1 or their ex vivo activation before myocardial delivery improves cell targeting to sites of injury, possibly providing a novel strategy for the management of the diseased heart.

Rationale: Two categories of cardiac stem cells (CSCs) with predominantly myogenic (mCSC) and vasculogenic (vCSC) properties have been characterized in the human heart. However, it is unknown whether functionally competent CSCs of both classes are present in the myocardium of patients affected by end-stage cardiac failure, and whether these cells can be harvested from relatively small myocardial samples., Objective: To establish whether a clinically relevant number of mCSCs and vCSCs can be isolated and expanded from endomyocardial biopsies of patients undergoing cardiac transplantation or left ventricular assist device implantation., Methods and Results: Endomyocardial biopsies were collected with a bioptome from the right side of the septum of explanted hearts or the apical LV core at the time of left ventricular assist device implantation. Two to 5 biopsies from each patient were enzymatically dissociated, and, after expansion, cells were sorted for c-kit (mCSCs) or c-kit and KDR (vCSCs) and characterized. mCSCs and vCSCs constituted 97% and 3% of the c-kit population, respectively. Population doubling time averaged 27 hours in mCSCs and vCSCs; 5×10(6) mCSCs and vCSCs were obtained in 28 and 41 days, respectively. Both CSC classes possessed significant growth reserve as documented by high telomerase activity and relatively long telomeres. mCSCs formed mostly cardiomyocytes, and vCSCs endothelial and smooth muscle cells., Conclusions: The growth properties of mCSCs and vCSCs isolated from endomyocardial biopsies from patients with advanced heart failure were comparable to those obtained previously from larger myocardial samples of patients undergoing elective cardiac surgery.

Background: Cardiac stem cells (CSCs) delivered to the infarcted heart generate a large number of small fetal-neonatal cardiomyocytes that fail to acquire the differentiated phenotype. However, the interaction of CSCs with postmitotic myocytes results in the formation of cells with adult characteristics., Methods and Results: On the basis of results of in vitro and in vivo assays, we report that the commitment of human CSCs (hCSCs) to the myocyte lineage and the generation of mature working cardiomyocytes are influenced by microRNA-499 (miR-499), which is barely detectable in hCSCs but is highly expressed in postmitotic human cardiomyocytes. miR-499 traverses gap junction channels and translocates to structurally coupled hCSCs favoring their differentiation into functionally competent cells. Expression of miR-499 in hCSCs represses the miR-499 target genes Sox6 and Rod1, enhancing cardiomyogenesis in vitro and after infarction in vivo. Although cardiac repair was detected in all cell-treated infarcted hearts, the aggregate volume of the regenerated myocyte mass and myocyte cell volume were greater in animals injected with hCSCs overexpressing miR-499. Treatment with hCSCs resulted in an improvement in ventricular function, consisting of a better preservation of developed pressure and positive and negative dP/dt after infarction. An additional positive effect on cardiac performance occurred with miR-499, pointing to enhanced myocyte differentiation/hypertrophy as the mechanism by which miR-499 potentiated the restoration of myocardial mass and function in the infarcted heart., Conclusions: The recognition that miR-499 promotes the differentiation of hCSCs into mechanically integrated cardiomyocytes has important clinical implications for the treatment of human heart failure.