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Although advances in rapid revascularization strategies following acute myocardial infarction (AMI) have led to improved short and long-term outcomes, the associated loss of cardiomyocytes and the subsequent remodeling result in an impaired ventricular function that can lead to heart failure or death. The poor regenerative capacity of the myocardium and the current lack of effective regenerative therapies have driven stem cell research in search of a possible solution. One approach involves the delivery of stem cells to the site of injury in order to stimulate repair response. Although animal studies initially delivered promising results, the application of similar techniques in humans has been hampered by poor target site retention and oncogenic considerations. In response, several alternative strategies, including the use of non-coding RNAs (ncRNAs), have been introduced with the aim of activating and regulating stem cells or inducing stem cell status in resident cells. Circular RNAs (circRNAs) and microRNAs (miRNAs) are ncRNAs with pivotal functions in cell proliferation and differentiation, whose role in stem cell regulation and potential significance for the field of cardiac regeneration is the primary focus of this review. We also address the general advantages of ncRNAs as promising drivers of cardiac regeneration and potent stem cell regulators.

Glutamine is a major energy source for rapidly dividing cells, such as hematopoietic stem cells and cancer cells. Reliance on glutamine is therefore regarded as a metabolic hallmark of proliferating cells. Moreover, reprogramming glutamine metabolism by various factors, including tissue type, microenvironment, pro-oncogenes, and tumor suppressor genes, can facilitate stem cell fate decisions, tumor recurrence, and drug resistance. However, the significance of glutamine metabolism in cardiomyocytes, an end-differentiated cell type, is not fully understood. Existing evidence suggests important roles of glutamine metabolism in the development of cardiovascular diseases. In this review, we have focused on glutaminolysis and its regulatory network in proliferating cells. We have summarized current findings about the role of glutamine utilization in cardiomyocytes and have discussed possibilities of targeting glutamine metabolism for the treatment of cardiovascular diseases., Competing Interests: Declaration of competing interest None., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The possibility of using stem cell-derived cardiomyocytes opens a new platform for modeling cardiac cell differentiation and disease or the development of new drugs. Progress in this field can be accelerated by high-throughput screening (HTS) technology combined with promoter reporter system. The goal of the study was to create and evaluate a responsive promoter reporter system that allows monitoring of iPSC differentiation towards cardiomyocytes. The lentiviral promoter reporter system was based on troponin 2 (TNNT2) and alpha cardiac actin (ACTC) with firefly luciferase and mCherry, respectively. The system was evaluated in two in vitro models. First, system followed the differentiation of TNNT2-luc-T2A-Puro-mCMV-GFP and hACTC-mcherry-WPRE-EF1-Neo from transduced iPSC line towards cardiomyocytes and revealed the significant decrease in both inserts copy number during the prolonged in vitro cell culture (confirmed by I-FISH, ddPCR, qPCR). Second, differentiated and contracting control cardiomyocytes (obtained from control non-reporter transduced iPSCs) were subsequently transduced with TNNT2-luc-T2A-Puro-CMV-GFP and hACTC-mcherry-WPRE-EF1-Neo lentiviruses to observe the functionality of obtained cardiomyocytes. Our results indicated that the reporter modified cell lines can be used for HTS applications, but it is essential to monitor the stability of the reporter sequence during extended cell in vitro culture.

Background: Cardiac stem cells (CSCs) exhibit age-dependent characteristics. Cited2 has been implicated in the regulation of heart development; however, there is little known about how Cited2 affects CSC aging., Results: Cited2 mRNA and protein level was downregulated in aging heart tissue and CSCs. Old (O)-CSCs showed decreased differentiation and proliferation capacities as compared to Young (Y)-CSCs, the decrease in cell proliferation, increase in apoptosis, and cell cycle arrest in G0/G1 phase in CSCs are mediated by knocdown CITED2expression in (Y)-CSCs., Conclusions: Cited2 plays an important role in cell cycle progression and in maintaining the balance between CSC proliferation and apoptosis in the process of aging without influencing cell fate decisions. These findings have important implications for cell-based therapies for heart repair.

During the development of cardiac hypertrophy, glucose deprivation (GD) associated with coronary microvascular rarefaction is caused, leading to cardiomyocyte death. Phosphorylation (inactivation) of eukaryotic elongation factor 2 (eEF2) by eEF2 kinase (eEF2K) inhibits protein translation, a highly energy consuming process, which plays protective roles against nutrient deprivation-induced cell death. We previously showed that eEF2 phosphorylation was increased in isolated heart from several cardiac hypertrophy models. In this study, we investigated whether eEF2K/eEF2 mediates the inhibition of cardiomyocyte death under GD condition. In H9c2 rat cardiomyoblasts cultured with serum-free medium, GD significantly augmented eEF2 phosphorylation and signals related to autophagy [increase of microtubule-associated protein 1 light chain 3 (LC3)-II to LC3-I ratio] and apoptosis (cleavage of caspase-3) as determined by Western blotting. GD induced cell death, which was augmented by eEF2K gene knockdown using a small interfering RNA. eEF2K gene knockdown significantly augmented GD-induced cleavage of caspase-3 and apoptotic nuclear condensation as determined by 4', 6-diamidino-2-phenylindole staining. In contrast, eEF2K gene knockdown significantly inhibited GD-induced increase of LC3-II to LC3-I ratio and autophagosome formation as determined by an immunofluorescence staining. An inhibitor of autophagy, 3-methyladenine or bafilomycin A1 significantly augmented GD-induced cleavage of caspase-3. Further, eEF2K gene knockdown significantly inhibited GD-induced phosphorylation of adenosine monophosphate-activated protein kinase (AMPK)α and its downstream substrate, unc-51 like autophagy activating kinase (ULK)1. An inhibitor of AMPK, dorsomorphin significantly inhibited GD-induced increase of LC3-II to LC3-I ratio. In conclusion, we for the first time revealed that eEF2K/eEF2 axis under GD condition mediates the inhibition of apoptotic H9c2 cell death at least in part via promotion of autophagy through AMPKα/ULK1 signaling pathway.

We propose a discrete in continuous mathematical model describing the in vitro growth process of biophsy-derived mammalian cardiac progenitor cells growing as clusters in the form of spheres (Cardiospheres). The approach is hybrid: discrete at cellular scale and continuous at molecular level. In the present model, cells are subject to the self-organizing collective dynamics mechanism and, additionally, they can proliferate and differentiate, also depending on stochastic processes. The two latter processes are triggered and regulated by chemical signals present in the environment. Numerical simulations show the structure and the development of the clustered progenitors and are in a good agreement with the results obtained from in vitro experiments.

In the heart, tissue-derived signals play a central role on recruiting/activating stem cell sources to induce cardiac lineage specification for maintenance of tissue homeostasis and repair. Cardiac resident stromal cells (CRSCs) may play a pivotal role in cardiac repair throughout their secretome. Here, we performed the characterization of CRSCs and their secretome by analyzing the composition of their culture-derived extracellular matrix (ECM) and conditioned medium (CM) and by investigating their potential effect on adipose-derived stem cell (ADSC) and progenitor cell behavior. We confirmed that CRSCs are a heterogeneous cell population whose secretome is composed by proteins related to cellular growth, immune response and cardiovascular development and function. We also observed that CRSC secretome was unable to change the behavior of ADSCs, except for proliferation. Additionally, CM from CRSCs demonstrated the potential to drive proliferation and cardiac differentiation of H9c2 cells and also the ability to induce angiogenesis in vitro. Our data suggest that the CRSCs can be a source of important modulating signals for cardiac progenitor cell recruitment/activation., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Efficient generation of cardiomyocytes from human pluripotent stem cells is critical for their regenerative applications. Microgravity and 3D culture can profoundly modulate cell proliferation and survival. Here, we engineered microscale progenitor cardiac spheres from human pluripotent stem cells and exposed the spheres to simulated microgravity using a random positioning machine for 3 days during their differentiation to cardiomyocytes. This process resulted in the production of highly enriched cardiomyocytes (99% purity) with high viability (90%) and expected functional properties, with a 1.5 to 4-fold higher yield of cardiomyocytes from each undifferentiated stem cell as compared with 3D-standard gravity culture. Increased induction, proliferation and viability of cardiac progenitors as well as up-regulation of genes associated with proliferation and survival at the early stage of differentiation were observed in the 3D culture under simulated microgravity. Therefore, a combination of 3D culture and simulated microgravity can be used to efficiently generate highly enriched cardiomyocytes.

Research has demonstrated that hypoxic preconditioning (HP) can enhance the survival and proliferation of cardiac progenitor cells (CPCs); however, the underlying mechanisms are not fully understood. Here, we report that HP of c-kit (+) CPCs inhibits p53 via the PI3K/Akt-DNMT1 pathway. First, CPCs were isolated from the hearts of C57BL/6 mice and further purified by magnetic-activated cell sorting. Next, these cells were cultured under either normoxia (H0) or HP for 6 hours (H6) followed by oxygen-serum deprivation for 24 hours (24h). Flow cytometric analysis and MTT assays revealed that hypoxia-preconditioned CPCs exhibited an increased survival rate. Western blot and quantitative real-time PCR assays showed that p53 was obviously inhibited, while DNMT1 and DNMT3β were both significantly up-regulated by HP. Bisulphite sequencing analysis indicated that DNMT1 and DNMT3β did not cause p53 promoter hypermethylation. A reporter gene assay and chromatin immunoprecipitation analysis further demonstrated that DNMT1 bound to the promoter locus of p53 in hypoxia-preconditioned CPCs. Together, these observations suggest that HP of CPCs could lead to p53 inhibition by up-regulating DNMT1 and DNMT3β, which does not result in p53 promoter hypermethylation, and that DNMT1 might directly repress p53, at least in part, by binding to the p53 promoter locus.

Background: Hyperglycemia is proposed to be an independent risk factor for cardiovascular morbidity and mortality. Preclinical studies suggest that diabetes mellitus exacerbates myocardial ischemia/reperfusion injury and attenuates the effects of cardioprotective strategies. The cardioprotective effects of postconditioning with the opioid analgesic remifentanil against ischemia/reperfusion injury under the hyperglycemic condition remain contradictory. Therefore, the aim of this study was to investigate the mechanisms by which hyperglycemia affects cardioprotection induced by remifentanil postconditioning., Materials and Methods: H9c2 cardiomyoblasts were cultured under the normoglycemic or hyperglycemic condition. Cells were exposed to hypoxia/reoxygenation (H/R) injury followed by hypoxia postconditioning (HPC group) or remifentanil postconditioning (RPC group). Cell viability, injury, and apoptosis were measured after each postconditioning treatment. Activation of endoplasmic reticulum stress (ERS) was analyzed by examining the protein levels of GRP78, CHOP, cleaved caspase-12 and cleaved caspase-3., Results: RPC significantly increased cell viability and reduced apoptosis in normoglycemic cardiomyoblasts, but not in hyperglycemic cardiomyoblasts. HPC and RPC markedly decreased the upregulation of GRP78, CHOP, cleaved caspase 12, and cleaved caspase 3 in response to H/R injury under the normoglycemic condition. Hyperglycemia significantly increased these ERS-associated biomarkers and apoptosis, which could not be reduced by HPC or RPC., Conclusions: Remifentanil postconditioning protected cardiomyoblasts from H/R injury under normoglycemia, at least in part, through inhibiting ERS-induced apoptosis. Hyperglycemia attenuated the cardioprotection conferred by remifentanil postconditioning, likely as a result of the exacerbated ERS. Inhibiting the ERS response may be an attractive strategy to enhance the cardioprotective effects of postconditioning in diabetic patients., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cardiomyocyte progenitor cells play essential roles in early heart development, which requires highly controlled cellular organization. microRNAs (miRs) are involved in various cell behaviors by post-transcriptional regulation of target genes. However, the roles of miRNAs in human cardiomyocyte progenitor cells (hCMPCs) remain to be elucidated. Our previous study showed that miR-134 was significantly downregulated in heart tissue suffering from congenital heart disease, underlying the potential role of miR-134 in cardiogenesis. In the present work, we showed that the upregulation of miR-134 reduced the proliferation of hCMPCs, as determined by EdU assay and Ki-67 immunostaining, while the inhibition of miR-134 exhibited an opposite effect. Both up- and downregulation of miR-134 expression altered the transcriptional level of cell-cycle genes. We identified Meis2 as the target of miR-134 in the regulation of hCMPC proliferation through bioinformatic prediction, luciferase reporter assay and western blot. The over-expression of Meis2 mitigated the effect of miR-134 on hCMPC proliferation. Moreover, miR-134 did not change the degree of hCMPC differentiation into cardiomyocytes in our model, suggesting that miR-134 is not required in this process. These findings reveal an essential role for miR-134 in cardiomyocyte progenitor cell biology and provide new insights into the physiology and pathology of cardiogenesis.

Stem cell-based therapy is currently tested in several trials of chronic heart failure. The main question is to determine how its implementation could be extended to common clinical practice. To fill this gap, it is critical to first validate the hypothesis that the grafted stem cells primarily act by harnessing endogenous repair pathways. The confirmation of this mechanism would have three major clinically relevant consequences: (i) the use of cardiac-committed cells, since even though cells primarily act in a paracrine manner, such a phenotype seems the most functionally effective; (ii) the optimization of early cell retention, rather than of sustained cell survival, so that the cells reside in the target tissue long enough to deliver the factors underpinning their action; and (iii) the reliance on allogeneic cells, the expected rejection of which should only have to be delayed since a permanent engraftment would no longer be the objective. One step further, the long-term objective of cell therapy could be to use the cells exclusively for producing factors and then to only administer them to the patient. The production process would then be closer to that of a biological pharmaceutic, thereby facilitating an extended clinical use., (© 2015 The Author(s).)

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.)

Aim: To study the effects of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-β-d-glucoside (THSG) on proliferation of rat cardiac stem cells (CSCs) in vitro., Materials and Methods: C-kit(+) cells were isolated from neonatal (1 day old) Sprague-Dawley rats by using flow cytometry. Optimal THSG treatment times and doses for growth of CSCs were analyzed. CSCs were treated with various THSG doses (0, 1, 10, and 100 μM) for 12h., Results: Sorted c-kit(+) cells exhibited self-renewing and clonogenic capabilities. Cell Counting Kit (CCK-8) and Proliferating Cell Nuclear Antigen (PCNA) ELISA test positive cells were significantly increased in THSG-treated groups compared with untreated controls. The percentage of S-phase cells also increased after THSG treatment. Moreover, we show that some c-kit(+) cells spontaneously express vascular endothelial growth factor (VEGF), T-box transcription factor (Tbx5), hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2), hyperpolarization-activated cyclic nucleotide gated 4 (HCN4), alpha myosin heavy chain (αMHC), and beta myosin heavy chain (βMHC) mRNA, and stem cell antigen 1 (Sca-1), cardiac troponin-I, GATA-4, Nkx2.5, and connexin 43 protein were also assessed in CSCs. However, their expression was significantly increased with THSG treatment when compared to untreated controls., Conclusion: THSG can increase proliferation of rat CSCs in vitro and thus, shows promise as a potential treatment strategy for stimulating endogenous stem cells to help repair the injured heart after myocardial infarction in patients., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Scl/Tal1 confers hemogenic competence and prevents ectopic cardiomyogenesis in embryonic endothelium by unknown mechanisms. We discovered that Scl binds to hematopoietic and cardiac enhancers that become epigenetically primed in multipotent cardiovascular mesoderm, to regulate the divergence of hematopoietic and cardiac lineages. Scl does not act as a pioneer factor but rather exploits a pre-established epigenetic landscape. As the blood lineage emerges, Scl binding and active epigenetic modifications are sustained in hematopoietic enhancers, whereas cardiac enhancers are decommissioned by removal of active epigenetic marks. Our data suggest that, rather than recruiting corepressors to enhancers, Scl prevents ectopic cardiogenesis by occupying enhancers that cardiac factors, such as Gata4 and Hand1, use for gene activation. Although hematopoietic Gata factors bind with Scl to both activated and repressed genes, they are dispensable for cardiac repression, but necessary for activating genes that enable hematopoietic stem/progenitor cell development. These results suggest that a unique subset of enhancers in lineage-specific genes that are accessible for regulators of opposing fates during the time of the fate decision provide a platform where the divergence of mutually exclusive fates is orchestrated., (© 2015 The Authors.)

Sulforaphane is a naturally occurring isothiocyanate capable of stimulating cellular antioxidant defenses and inducing phase 2 detoxifying enzymes, which can protect cells against oxidative damage. Oxidative stress and apoptosis are intimately involved in the pathophysiology of cardiac diseases. Although sulforaphane is known for its anticancer benefits, its role in cardiac cells is just emerging. The aim of the present study was to investigate whether sulforaphane can modulate oxidative stress, apoptosis, and correlate with PGC-1α, a transcriptional cofactor involved in energy metabolism. H9c2 cardiac myoblasts were incubated with R-sulforaphane 5 µmol/L for 24 h. Cell viability, ANP gene expression, oxidative stress and apoptosis markers, and protein expression of PGC-1α were studied. In cells treated with sulforaphane, cellular viability increased (12 %) and ANP gene expression decreased (46 %) compared to control cells. Moreover, sulforaphane induced a significant increase in superoxide dismutase (103 %), catalase (101 %), and glutathione S-transferase (72 %) activity, reduced reactive oxygen species levels (15 %) and lipid peroxidation (65 %), as well as stimulated the expression of the cytoprotective enzyme heme oxygenase-1 (4-fold). Sulforaphane also promoted an increase in the expression of the anti-apoptotic protein Bcl-2 (60 %), decreasing the Bax/Bcl-2 ratio. Active Caspase 3\7 and p-JNK/JNK were also reduced by sulforaphane, suggesting a reduction in apoptotic signaling. This was associated with an increased protein expression of PGC-1α (42 %). These results suggest that sulforaphane offers cytoprotection to cardiac cells by activating PGC1-α, reducing oxidative stress, and decreasing apoptosis signaling.

Over the past 2 decades, cardiac regeneration has evolved from an exotic fringe of cardiovascular biology to the forefront of molecular, genetic, epigenetic, translational, and clinical investigations. The unmet patient need is the paucity of self-repair following infarction. Robust regeneration seen in models such as zebrafish and newborn mice has inspired the field, along with encouragement from modern methods that make even low levels of restorative growth discernible, changing the scientific and technical landscape for effective counter-measures. Approaches under study to augment cardiac repair complement each other, and encompass grafting cells of diverse kinds, restarting the cell cycle in post-mitotic ventricular myocytes, reprogramming non-myocytes, and exploiting the dormant progenitor/stem cells that lurk within the adult heart. The latter are the emphasis of the present review. Cardiac-resident stem cells (CSC) can be harvested from heart tissue, expanded, and delivered to the myocardium as a therapeutic product, whose benefits may be hoped to surpass those achieved in human trials of bone marrow. However, important questions are prompted by such cells' discovery. How do they benefit recipient hearts? Do they contribute, measurably, as an endogenous population, to self-repair? Even if "no," might CSCs be targets for activation in situ by growth factors and other developmental catalysts? And, what combination of distinguishing markers best demarcates the cells with robust clonal growth and cardiogenic potential?

Aims: Our previous study found that A83-01, a small molecule type 1 TGFβ receptor inhibitor, could induce proliferation of postnatal Nkx2.5(+) cardiomyoblasts in vitro and enhance their cardiomyogenic differentiation. The present study addresses whether A83-01 treatment in vivo could increase cardiomyogenesis and improve cardiac function after myocardial infarction through an Nkx2.5(+) cardiomyoblast-dependent process., Methods and Results: To determine the effect of A83-01 on the number of Nkx2.5(+) cardiomyoblasts in the heart after myocardial injury, we treated transgenic Nkx2.5 enhancer-GFP reporter mice for 7 days with either A83-01 or DMSO and measured the number of GFP(+) cardiomyoblasts in the heart at 1 week after injury by flow cytometry. To determine the degree of new cardiomyocyte formation after myocardial injury and the effect of A83-01 in this process, we employed inducible Nkx2.5 enhancer-Cre transgenic mice to lineage label postnatal Nkx2.5(+) cardiomyoblasts and their differentiated progenies after myocardial injury. We also examined the cardiac function of each animal by intracardiac haemodynamic measurements. We found that A83-01 treatment significantly increased the number of Nkx2.5(+) cardiomyoblasts at baseline and after myocardial injury, resulting in an increase in newly formed cardiomyocytes. Finally, we showed that A83-01 treatment significantly improved ventricular elastance and stroke work, leading to improved contractility after injury., Conclusion: Pharmacological inhibition of TGFβ signalling improved cardiac function in injured mice and promoted the expansion and cardiomyogenic differentiation of Nkx2.5(+) cardiomyoblasts. Direct modulation of resident cardiomyoblasts in vivo may be a promising strategy to enhance therapeutic cardiac regeneration., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

The Drosophila heart is a linear organ formed by the movement of bilaterally specified progenitor cells to the midline and adherence of contralateral heart cells. This movement occurs through the attachment of heart cells to the overlying ectoderm which is undergoing dorsal closure. Therefore heart cells are thought to move to the midline passively. Through live imaging experiments and analysis of mutants that affect the speed of dorsal closure we show that heart cells in Drosophila are autonomously migratory and part of their movement to the midline is independent of the ectoderm. This means that heart formation in flies is more similar to that in vertebrates than previously thought. We also show that defects in dorsal closure can result in failure of the amnioserosa to properly degenerate, which can physically hinder joining of contralateral heart cells leading to a broken heart phenotype., (Copyright © 2014 Elsevier Inc. All rights reserved.)

To form the primary heart tube in zebrafish, bilateral cardiac precursor cells (CPCs) migrate toward the midline beneath the endoderm. Mutants lacking endoderm and fish with defective sphingosine 1-phosphate (S1P) signaling exhibit cardia bifida. Endoderm defects lead to the lack of foothold for the CPCs, whereas the cause of cardia bifida in S1P signaling mutants remains unclear. Here we show that S1P signaling regulates CPC migration through Yes-associated protein 1 (Yap1)-dependent endoderm survival. Cardia bifida seen in spns2 (S1P transporter) morphants and s1pr2 (S1P receptor-2) morphants could be rescued by endodermal expression of nuclear localized form of yap1. yap1 morphants had decreased expression of the Yap1/Tead target connective tissue growth factor a (Ctgfa) and consequently increased endodermal cell apoptosis. Consistently, ctgfa morphants showed defects of the endodermal sheet and cardia bifida. Collectively, we show that S1pr2/Yap1-regulated ctgfa expression is essential for the proper endoderm formation required for CPC migration., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cell proliferation from pre-existing cardiomyocytes is a major source of cells for normal mammalian myocardial renewal or for regeneration after myocardial injury. These proliferative cardiomyocytes may act differently from the postmitotic cardiomyocytes in a stressed heart. Extracellular matrix molecule CCN1 is produced to promote Fas ligand (FasL)-induced cardiomyocyte apoptosis in mice with stress-induced cardiac injury. We aimed to investigate the effect of CCN1 on the proliferative cardiomyocytes. We used rat embryonic cardiomyoblast H9c2 cells to study the cardiotoxicity of CCN1. We found that FasL dose-dependently increased the X-linked inhibitor of apoptosis protein (XIAP) levels to prevent the progression of apoptosis in H9c2 cells. CCN1, though it did not induce apoptosis by itself, sensitized H9c2 cells to FasL-induced apoptosis. CCN1 functions by engaging its cell-surface receptor integrin α6β1 and elevating reactive oxygen species levels, which leads to mitogen-activated protein kinase p38 activation, cytosolic Bax translocation to mitochondria, and the release of mitochondrial Smac and HtrA2 to cytosol. These elevated cytosolic Smac and HtrA2 dismantle the inhibition of XIAP, thereby facilitating the activation of caspase-3 and the apoptosis-induced by FasL. In summary, we demonstrated a novel mechanism underlying the resistance of cardiomyoblasts to FasL-induced apoptosis, and the pro-apoptotic function of CCN1 by disrupting this resistance., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The use of triple systemic transplantation of cardiomyoblasts raised from the culture of allogenic bone marrow mesenchymal stem cells of a healthy donor according to the new medical technology licensed by Federal Service on Surveillance in Healthcare in the therapy of a patient with late radiation cardiomyopathy and radiation exudative pericarditis developed 45 years after radiation therapy for Hodgkin lymphoma. High efficiency of systemic transplantation of mesenchymal stem cells partially differentiated towards cardiomyocytes was demonstrated. The therapeutic effect persists for more than 2 years. Possible mechanisms of the therapeutic effect of this type of stem cells and the prospects of using cell therapy in the treatment of late radiation injuries of vital organs and tissues are discussed.

Cardiovascular diseases (CVD) are a global epidemic in developed countries. Cumulative evidence suggests that myocyte formation is preserved during postnatal life, in adulthood or senescence, suggesting the existence of a growth reserve of the heart throughout lifespan. Several medical therapeutic approaches to CVD have considerably improved the clinical outcome for patients. Intense interest has been focused on regenerative medicine as an emerging strategy for CVD. Cellular therapeutic approaches have been proposed for enhancing survival and propagation of stem cells in myocardium, leading to cardiac cellular repair. Strong epidemiological and clinical data exists concerning the impact of regular physical exercise on cardiovascular health. Several mechanisms of acute and chronic exercise-induced cardiovascular adaptations to exercise have been presented, considering primary and secondary prevention of CVD. In this context, exercise-related improvements in the function and regeneration of the cardiovascular system may be associated with the exercise-induced activation, mobilization, differentiation, and homing of stem and progenitor cells. In this review several topics will be addressed concerning the relation between exercise, recruitment and biological activity of blood-circulating progenitor cells and resident cardiac stem cells. We hypothesize that exercise-induced stem cell activation may enhance overall heart function and improve the efficacy of cardiac cellular therapeutic protocols., (© Georg Thieme Verlag KG Stuttgart · New York.)

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.

A potent analog (HNG) of the endogenous peptide humanin protects against myocardial ischemia-reperfusion (MI-R) injury in vivo, decreasing infarct size and improving cardiac function. Since oxidative stress contributes to the damage from MI-R we tested the hypotheses that: (1) HNG offers cardioprotection through activation of antioxidant defense mechanisms leading to preservation of mitochondrial structure and that, (2) the activity of either of a pair of non-receptor tyrosine kinases, c-Abl and Arg is required for this protection. Rat cardiac myoblasts (H9C2 cells) were exposed to nanomolar concentrations of HNG and to hydrogen peroxide (H2O2). Cells treated with HNG in the presence of H2O2 demonstrated reduced intracellular reactive oxygen species (ROS), preserved mitochondrial membrane potential, ATP levels and mitochondrial structure. HNG induced activation of catalase and glutathione peroxidase (GPx) within 5 min and decreased the ratio of oxidized to reduced glutathione within 30 min. siRNA knockdown of both Abl and Arg, but neither alone, abolished the HNG-mediated reduction of ROS in myoblasts exposed to H2O2. These findings demonstrate an HNG-mediated, Abl- and Arg-dependent, rapid and sustained activation of critical cellular defense systems and attenuation of oxidative stress, providing mechanistic insights into the observed HNG-mediated cardioprotection in vivo., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aims: The study was designed to evaluate the mechanisms of cardiac regeneration after injury and to determine how to restore that capacity in aged individuals. The adult heart retains a small population of nascent cells that have myeloid, mesenchymal, and mesodermal capabilities, which play an essential role in the recovery of ventricular function after injury. In aged individuals, these cells are diminished and dysfunctional. We evaluated the derivation of some of these cardiac progenitors and a method to restore their number and function., Methods and Results: We first demonstrated that aged mice have fewer progenitors in both the bone marrow (BM) and the myocardium, which correlated with the extent of cardiac dysfunction after injury. Bone marrow chimerism established in aged mice with young BM donors restored both myocardial progenitors and cardiac function, but neither was restored with aged BM donors. Cardiac micro-chimerism in aged mice was established with young BM cells, which restored cardiac function after injury, even with old peripheral BM cells. The young cardiac-resident BM-derived progenitor cells in the aged myocardium persisted for at least a year, and after myocardial infarction they actively proliferated and enhanced cardiac repair through paracrine mechanisms., Conclusion: Bone marrow reconstitution with young BM cells in aged recipients restored progenitors in both the BM and, most importantly, the myocardium. The number and function of cardiac-resident BM-derived progenitor cells in the aged myocardium prior to injury was the major determinant for successful recovery of cardiac function. The aged heart was rejuvenated with young BM cells.

To evaluate potential roles of nitric oxide (NO) in the regulation of the endothelial lineage and neovascular processes (vasculogenesis and angiogenesis) we evaluated endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (p-eNOS) expression in 7.2-8.5 days post-coitum (dpc) mouse embryos. Analysis revealed that p-eNOS((S1177)) but not P-eNOS((S617)) or P-eNOS((T495)) was expressed in a subpopulation of angioblasts (TAL-1(+)/Flk-1(+)/CD31(-)/CD34(-)/VE-Cadherin(-)) at 7.2 dpc. A role of the VEGF/Akt1/eNOS signaling pathway in the regulation of the endothelial cell (EC) lineage was suggested by the strong correlation observed between cell division and p-eNOS((S1177)) expression in both angioblasts and embryonic endothelial cells (EECs, TAL-1(+)/Flk-1(+)/CD31(+)/CD34(+)/VE-Cadherin(+)). Our studies using Akt1 null mouse embryos show a reduction in p-eNOS((S1177)) expression in angioblast and EECs that is correlated with a decrease in endothelial cell proliferation and results in changes in VEGF-induced vascular patterning. Further, we show that VEGF-mediated cell proliferation in Flk-1(+) cells in allantoic cultures is decreased by pharmacological inhibitors of the VEGF/Akt1/eNOS signaling pathways. Taken together, our findings suggest that VEGF-mediated eNOS phosphorylation on Ser1177 regulates angioblast and EEC division, which underlies the formation of blood vessels and vascular networks., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The metabolic syndrome (MetS) is a constellation of multiple metabolic risk factors including obesity, glucose intolerance, insulin resistance, dyslipidemia and hypertension. Individuals with MetS are found to be afflicted with an increased risk of type 2 diabetes mellitus and overall cardiovascular diseases. One of the common comorbidities of MetS is the impairment of heart function en route to loss of cardiomyocytes and ultimately heart failure. Although it is accepted that cardiomyocytes are terminally differentiated, recent evidence has challenged this concept to indicate the ability of cardiomyocytes to regenerate from progenitor cells of the heart and other organs. Moreover, it has been suggested that pathological conditions such as MetS may play a role in the regulation of cardiomyocyte regeneration. This mini-review will discuss the role of MetS in the regulatory machineries of cardiomyocyte regeneration.

A characteristic component of the anti-neoplastic doxorubicin (DOX)-induced cardiac toxicity is the delayed and persistent toxicity, with cancer childhood survivors developing cardiac failure later in life. The mechanisms behind this persistent toxicity are unknown, although one of the consequences of early childhood treatment with DOX is a specific removal of cardiac progenitor cells. DOX treatment may be more toxic to undifferentiated muscle cells, contributing to impaired cardiac development and toxicity persistence. H9c2 myoblasts, a rat embryonic cell line, which has the ability to differentiate into a skeletal or cardiac muscle phenotype, can be instrumental in understanding DOX cytotoxicity in different differentiation stages. H9c2 cell differentiation results in decreased cell proliferation and increased expression of a differentiated muscle marker. Differentiated H9c2 cells accumulated more DOX and were more susceptible to DOX-induced cytotoxicity. Differentiated cells had increased levels of mitochondrial superoxide dismutase and Bcl-xL, an anti-apoptotic protein. Of critical importance for the mechanisms of DOX toxicity, p53 appeared to be equally activated regardless of the differentiation state. We suggest that although more differentiated H9c2 muscle cells appear to have more basal mechanisms that would predict higher protection, DOX toxicity is higher in the differentiated population. The results are instrumental in the understanding of stress responses of this specific cell line in different differentiation stages to the cardiotoxicity caused by anthracyclines.

Unique insights for the reprograming of cell lineages have come from embryonic development in the ascidian Ciona, which is dependent upon the transcription factors Ci-ets1/2 and Ci-mesp to generate cardiac progenitors. We tested the idea that mammalian v-ets erythroblastosis virus E26 oncogene homolog 2 (ETS2) and mesoderm posterior (MESP) homolog may be used to convert human dermal fibroblasts into cardiac progenitors. Here we show that murine ETS2 has a critical role in directing cardiac progenitors during cardiopoiesis in embryonic stem cells. We then use lentivirus-mediated forced expression of human ETS2 to convert normal human dermal fibroblasts into replicative cells expressing the cardiac mesoderm marker KDR(+). However, although neither ETS2 nor the purported cardiac master regulator MESP1 can by themselves generate cardiac progenitors de novo from fibroblasts, forced coexpression of ETS2 and MESP1 or cell treatment with purified proteins reprograms fibroblasts into cardiac progenitors, as shown by the de novo appearance of core cardiac transcription factors, Ca(2+) transients, and sarcomeres. Our data indicate that ETS2 and MESP1 play important roles in a genetic network that governs cardiopoiesis.

Alcohol exposure during pregnancy may cause congenital heart disease (CHD). In our previous studies, we found that alcohol selectively increased acetylation of histone H3 at lysine 9 (H3K9) and enhanced the expression of heart development-related genes in cardiac progenitor cells. The objective of this study is to investigate the protective effects of histone acetyltransferases (HATs) inhibitor, curcumin, on histone hyperacetylation and the over-expression of heart development genes induced by alcohol. Western blot analysis was employed to detect the acetylation levels of histone H3K9 and real-time PCR was applied to measure the expressions of heart development-related transcription factors, GATA4, Mef2c and Tbx5 (GMT). Our results showed that alcohol increased the acetylation of H3K9 by 2.76-fold (P<0.05) and significantly enhanced the expression of GATA4 and Mef2c (P<0.05). When cells were treated with alcohol plus 25 μM curcumin, the hyperacetylation of H3K9 and over-expression of GATA4 and Mef2c by alcohol was reversed. These data indicate that curcumin can correct the over-expression of cardiac genes by reversing the alcohol induced hyperacetylation of histone H3 at lysine 9 in cardiac progenitor cells, suggesting that curcumin is protective against alcohol-induced cardiac gene over-expression that may result in heart malformations., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The cardiotoxicity of cyclosporine A (CsA) limits its clinical application in extensive and long-term therapies. Our group has shown that CsA induces myocardium cell apoptosis in vivo and increases calcium-sensing receptor (CaSR) expression. However, its molecular mechanism remains unknown. The purpose of this study was to determine whether CaSR plays an essential role in CsA-induced apoptosis in H9c2 cells and to investigate the role of the mitogen-activated protein kinase (MAPK) signaling cascade in this process. H9c2 cells were treated with CsA in a dose-dependent manner, and decreased Bcl-2 expression, increased Bax expression, and caspase-3 activation were observed. In a time-dependent manner, CsA increased CaSR expression, activated the extracellularly regulated kinase (ERK) and p38 MAPK pathways, and inactivated the c-Jun N-terminal kinase (JNK) MAPK signaling pathway. When H9c2 cardiomyoblast cells pretreated with gadolinium chloride (GdCl(3)), a CaSR activator, were treated with CsA, decreased phosphorylation of ERK1/2, increased phosphorylation of p38, decreased Bcl-2 expression, increased Bax expression, and activated caspase-3 were observed. Cells pretreated with the CaSR inhibitor NPS2390 inhibited this process. Furthermore, the MEK1/2 inhibitor U0126 and the p38 MAPK inhibitor SB203580 markedly blocked the effect of CsA on cell apoptosis, apoptotic-related protein expression, and caspase-3 activation. These findings showed that CsA induced apoptosis in H9c2 cells in vitro, and CaSR mediated the degradation of ERK MAPK and the upregulation of the p38 MAPK pathway involved in CsA-induced H9c2 cardiomyoblast cell apoptosis.

The ErbB2 receptor is a proto-oncogene associated with a poor prognosis in breast cancer. Herceptin, the only humanized anti-ErbB2 antibody currently in clinical use, has proven to be an essential tool in the immunotherapy of breast carcinoma, but induces cardiotoxicity. ErbB2 is involved in the growth and survival pathway of adult cardiomyocytes; however, its levels in the adult heart are much lower than those found in breast cancer cells, the intended targets of anti-ErbB2 antibodies. Furthermore, clinical trials have shown relatively low cardiotoxicity for Lapatinib, a dual kinase inhibitor of EGFR and ErbB2, and Pertuzumab, a new anti-ErbB2 monoclonal antibody currently in clinical trials, which recognizes an epitope distant from that of Herceptin. A novel human antitumor compact anti-ErbB2 antibody, Erb-hcAb, selectively cytotoxic for ErbB2-positive cancer cells in vitro and vivo, recognizes an epitope different from that of Herceptin, and does not show cardiotoxic effects both in vitro on rat and human cardiomyocytes and in vivo on a mouse model. We investigated the molecular basis of the different cardiotoxic effects among the ErbB2 inhibitors by testing their effects on the formation of the Neuregulin 1β (NRG-1)/ErbB2/ErbB4 complex and on the activation of its downstream signaling. We report herein that Erb-hcAb at difference with Herceptin, 2C4 (Pertuzumab) and Lapatinib, does not affect the ErbB2-ErbB4 signaling pathway activated by NRG-1 in cardiac cells. These findings may have important implications for the mechanism and treatment of anti-ErbB2-induced cardiotoxicity.

While recent data have supported the capacity for a neonatal heart to undergo cardiomyogenesis, it is unclear whether these new cardiomyocytes arise from an immature cardiomyoblast population or from the division of mature cardiomyocytes. By following the expression of enhanced Green Fluorescent Protein (eGFP) in an Nkx2.5 enhancer-eGFP transgenic mice, we have identified a population of immature cells that can undergo cardiomyogenic as well as smooth muscle cell differentiation in the neonatal heart. Here, we examined growth factors and small molecule regulators that potentially regulate the proliferation and cardiomyogenic versus smooth muscle cell differentiation of neonatal Nkx2.5-GFP (+) cells in vitro. We found that A83-01 (A83), an inhibitor of TGF-βRI, was able to induce an expansion of neonatal Nkx2.5-eGFP (+) cells. In addition, the ability of A83 to expand eGFP (+) cells in culture was dependent on signalling from the mitogen-activated protein kinase kinase (MEK) as treatment with a MEK inhibitor, PD0325901, abolished this effect. On the other hand, activation of neonatal Nkx2.5-eGFP (+) cells with TGF-β1, but not activin A nor BMP2, led to smooth muscle cell differentiation, an effect that can be reversed by treatment with A83. In summary, small molecule inhibition of TGF-β signalling may be a promising strategy to induce the expansion of a rare population of postnatal cardiomyoblasts., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Background: The formation of a tubular organ, such as the heart, requires the communication of positional and polarity signals between migratory cells. Key to this process is the establishment of a new luminal domain on the cell surface, generally from the apical domain of a migratory cell. This domain will also acquire basal properties, as it will produce a luminal extracellular matrix. Integrin receptors are the primary means of cell adhesion and adhesion signaling with the extracellular matrix. Here we characterise the requirement of Integrins in a genetic model of vasculogenesis, the formation of the heart in Drosophila., Results: As with vertebrates, the Drosophila heart arises from lateral mesoderm that migrates medially to meet their contralateral partners, to then assemble a midline vessel. During migration, Integrins are among the first proteins restricted to the presumptive luminal domain of cardioblasts. Integrins are required for normal levels of leading edge membrane motility. Apical accumulation of Integrins is enhanced by Robo, and reciprocally, apicalisation of luminal factors like Slit and Robo requires Integrin function. Integrins may provide a template for the formation of a lumen by stabilising lumen factors like Robo. Subsequent to migration, Integrin is required for normal cardioblast alignment and lumen formation. This phenotype is most readily modified by other mutations that affect adhesion, such as Talin and extracellular matrix ligands., Conclusion: Our findings reveal an instructive role for Integrins in communicating polarising information to cells during migration, and during transition to an epithelial tube structure.

Background: The cardiac progenitor cells for the outflow tract (OFT) reside in the visceral mesoderm and mesodermal core of the pharyngeal region, which are defined as the secondary and anterior heart fields (SHF and AHF), respectively., Results: Using chick embryos, we injected fluorescent-dye into the SHF or AHF at stage 14, and the destinations of the labeled cells were examined at stage 31. Labeled cells from the right SHF were found in the myocardium on the left dorsal side of the OFT, and cells from the left SHF were detected on the right ventral side of the OFT. Labeled cells from the right and left AHF migrated to regions of the ventral wall of the OFT close to the aortic and pulmonary valves, respectively., Conclusion: These observations indicate that myocardial progenitors from the SHF and AHF contribute to distinct conotruncal regions and that cells from the SHF migrate rotationally while cells from the AHF migrate in a non-rotational manner., (Copyright © 2011 Wiley Periodicals, Inc.)

Exercise training fosters the health and performance of the cardiovascular system, and represents nowadays a powerful tool for cardiovascular therapy. Exercise exerts its beneficial effects through reducing cardiovascular risk factors, and directly affecting the cellular and molecular remodelling of the heart. Traditionally, moderate endurance exercise training has been viewed to determine a balanced and revertible physiological growth, through cardiomyocyte hypertrophy accompanied by appropriate neoangiogenesis (the Athlete's Heart). These cellular adaptations are due to the activation of signalling pathways and in particular, the IGF-1/IGF-1R/Akt axis appears to have a major role. Recently, it has been shown that physical exercise determines cardiac growth also through new cardiomyocyte formation. Accordingly, burgeoning evidence indicates that exercise training activates circulating, as well as resident tissue-specific cardiac, stem/progenitor cells. Dissecting the mechanisms for stem/progenitor cell activation with exercise will be instrumental to devise new effective therapies, encompassing myocardial regeneration for a large spectrum of cardiovascular diseases.

Objective: To investigate the effects of exogenous basic fibroblast growth factor (bFGF) on myocardial regeneration after acute myocardial infarction (AMI)., Methods: AMI models were established by ligating the mid-third of left anterior descending artery, thereafter, miniswines were randomly divided into control (none treatment, n = 6) and bFGF groups (n = 6). For the bFGF group, bFGF (100 μg) was injected with a sterile microinjection at five sites within the ischemic region. 5-Bromo-2-deoxyuridine (250 mg) was administrated intravenously twice a week after the operation, to label cells undergoing DNA replication. The expression of stromal cell-derived factor-1α (SDF-1α) and CXC chemokine receptor 4 (CXCR4), cardiac stem cell-mediated myocardial regeneration, myocardial apoptosis, histological and immunohistochemical analyses, and cardiac function were evaluated at different time points., Results: Four weeks after bFGF therapy, it showed an increased vessel density and myocardial perfusion (P < 0.001), upregulative expression of SDF-1α and CXCR4 (P < 0.001), increased c-kit and 5-bromo-2-deoxyuridine-positive cells (P < 0.001), enhanced myocardial viability (P < 0.001), and improved left ventricular ejection fraction (P = 0.007), compared with the control., Conclusion: Exogenous bFGF was shown to have increased angiogenesis and myocardial perfusion, promoted myocardial regeneration by activating the SDF-1α/CXCR4 axis, and thereby improved the cardiac function, which may provide a new therapeutic strategy for AMI.

In the past, clinical trials transplanting bone marrow-derived mononuclear cells reported a limited improvement in cardiac function. Therefore, the search for stem cells leading to more successful stem cell therapies continues. Good candidates are the so-called cardiac stem cells (CSCs). To date, there is no clear evidence to show if these cells are intrinsic stem cells from the heart or mobilized cells from bone marrow. In this study we performed a comparative study between human mesenchymal stem cells (hMSCs), purified c-kit(+) CSCs, and cardiosphere-derived cells (CDCs). Our results showed that hMSCs can be discriminated from CSCs by their differentiation capacity towards adipocytes and osteocytes and the expression of CD140b. On the other hand, cardiac progenitors display a greater cardiomyogenic differentiation capacity. Despite a different isolation protocol, no distinction could be made between c-kit(+) CSCs and CDCs, indicating that they probably derive from the same precursor or even are the same cells.

Background: Human cardiac progenitor cells (hCPCs) may promote myocardial regeneration in adult ischemic myocardium. The regenerative capacity of hCPCs in young patients with nonischemic congenital heart defects for potential use in congenital heart defect repair warrants exploration., Methods and Results: Human right atrial specimens were obtained during routine congenital cardiac surgery across 3 groups: neonates (age, <30 days), infants (age, 1 month to 2 years), and children (age, >2 to ≤13 years). C-kit(+) hCPCs were 3-fold higher in neonates than in children >2 years of age. hCPC proliferation was greatest during the neonatal period as evidenced by c-kit(+) Ki67(+) expression but decreased with age. hCPC differentiation capacity was also greatest in neonatal right atrium as evidenced by c-kit(+), NKX2-5(+), NOTCH1(+), and NUMB(+) expression. Despite the age-dependent decline in resident hCPCs, we isolated and expanded right atrium-derived CPCs from all patients (n=103) across all ages and diagnoses using the cardiosphere method. Intact cardiospheres contained a mix of heart-derived cell subpopulations that included cardiac progenitor cells expressing c-kit(+), Islet-1, and supporting cells. The number of c-kit(+)-expressing cells was highest in human cardiosphere-derived cells (hCDCs) grown from neonatal and infant right atrium. Furthermore, hCDCs could differentiate into diverse cardiovascular lineages by in vitro differentiation assays. Transplanted hCDCs promoted greater myocardial regeneration and functional improvement in infarcted myocardium than transplanted cardiac fibroblasts., Conclusions: Resident hCPCs are most abundant in the neonatal period and rapidly decrease over time. hCDCs can be reproducibly isolated and expanded from young human myocardial samples regardless of age or diagnosis. hCPCs are functional and have potential in congenital cardiac repair.

The advent of stem cell therapy brings about the hope to restore the loss of cardiac pacemaker cells. However, it is largely unknown whether cardiac stem cells are able to differentiate into pacemaker cells. The purpose of this study was to determine whether the heart of large juvenile mammals contains cardiac stem cells (CSCs), which are suitable as seed cells for restoration of cardiac pacemaker cell. The c-kit(+) CSCs were isolated from one-month-old mongrel dogs. CSCs that we sorted were self-renewing, and they could proliferate by clonal expansion. CSCs could differentiate into cardiac muscle, smooth muscle and endothelial cells at rates of 10.5 ± 4.2%, 13.5 ± 5.1% and 12.9 ± 3.5%, respectively, at week 4, as judged by the expression of respective differentiation markers: cardiac troponin I, smooth muscle actin, and CD31. At week 8, the differentiation rates were further increased to 23.2 ± 3.6%, 25.9 ± 6.6% and 28.3 ± 6.1% (P < 0.05 for each marker). Some of cells derived from CSCs could express cardiac transcription factor GATA-4 after week 2 and express pacing-related genes, including hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) and HCN4 after week 4. Importantly, a fraction of CSCs demonstrated the presence of inward currents that indicate the expression of inward current channels. In conclusion, c-kit(+) CSCs may differentiate into cardiac muscle cell and sinus node-like cells, suggesting that CSCs would be useful as seed cells in treating sinus bradycardiac disorders or exploring the mechanism of pacemaker activity.

Myocardial infarction resulting in irreversible loss of cardiomyocytes (CMs) remains a leading cause of heart failure. Although cell transplantation has modestly improved cardiac function, major challenges including increasing cell survival, engraftment, and functional integration with host tissue, remain. Embryonic stem cells (ESCs), which can be differentiated into cardiac progenitors (CPs) and CMs, represent a candidate cell source for cardiac cell therapy. However, it is not known what specific cell type or condition is the most appropriate for transplantation. This problem is exasperated by the lack of efficient and predictive strategies to screen the large numbers of parameters that may impact cell transplantation. We used a cardiac tissue model, engineered heart tissue (EHT), and quantitative molecular and electrophysiological analyses, to test transplantation conditions and specific cell populations for their potential to functionally integrate with the host tissue. In this study, we validated our analytical platform using contractile mouse neonatal CMs (nCMs) and noncontractile cardiac fibroblasts (cFBs), and screened for the integration potential of ESC-derived CMs and CPs (ESC-CMs and -CPs). Consistent with previous in vivo studies, cFB injection interfered with electrical signal propagation, whereas injected nCMs improved tissue function. Purified bioreactor-generated ESC-CMs exhibited a diminished capacity for electrophysiological integration; a result correlated with lower (compared with nCMs) connexin 43 expression. ESC-CPs, however, appeared able to appropriately mature and integrate into EHT, enhancing the amplitude of tissue contraction. Our results support the use of EHT as a model system to accelerate development of cardiac cell therapy strategies.

The potential of genetically modified cardiomyoblasts in treating damaged myocardium is well known. However, efficient delivery of these cells is of major concern during treatment. The limiting factors are the massive cell death that occurs soon after their intramyocardial transplantation into the beating heart. To address these problems, we generated recombinant baculoviruses (BacMam viruses) which efficiently transduced cardiomyoblast cells under optimized conditions. These genetically modified cells were then protected in a new polymeric microcapsule using poly-ethylene-glycol (PEG), alginate, and poly-L-lysine (PLL) polymers for efficient delivery. Results showed that microcapsules maintain cell viability and support cell proliferation for at least 30 days. The capsules exhibit strong immunoprotective potential and have high mechanical and osmotic stability with more than 70% intact capsules. The encased transduced cells showed a rapid transgene expression inside the capsule for at least 15 days. However, preclinical studies are needed to further explore its long-term functional benefits.

We analyzed the effect of conditional, alphaMHC-dependent genetic beta-catenin depletion and stabilization on cardiac remodeling following experimental infarct. beta-Catenin depletion significantly improved 4-week survival and left ventricular (LV) function (fractional shortening: CT(Deltaex3-6): 24 +/- 1.9%; beta-cat(Deltaex3-6): 30.2 +/- 1.6%, P < 0.001). beta-Catenin stabilization had opposite effects. No significant changes in adult cardiomyocyte survival or hypertrophy were observed in either transgenic line. Associated with the functional improvement, LV scar cellularity was altered: beta-catenin-depleted mice showed a marked subendocardial and subepicardial layer of small cTnT(pos) cardiomyocytes associated with increased expression of cardiac lineage markers Tbx5 and GATA4. Using a Cre-dependent lacZ reporter gene, we identified a noncardiomyocyte cell population affected by alphaMHC-driven gene recombination localized to these tissue compartments at baseline. These cells were found to be cardiac progenitor cells since they coexpressed markers of proliferation (Ki67) and the cardiomyocyte lineage (alphaMHC, GATA4, Tbx5) but not cardiac Troponin T (cTnT). The cell population overlaps in part with both the previously described c-kit(pos) and stem cell antigen-1 (Sca-1)(pos) precursor cell population but not with the Islet-1(pos) precursor cell pool. An in vitro coculture assay of highly enriched (>95%) Sca-1(pos) cardiac precursor cells from beta-catenin-depleted mice compared to cells isolated from control littermate demonstrated increased differentiation toward alpha-actin(pos) and cTnT(pos) cardiomyocytes after 10 days (CT(Deltaex3-6): 38.0 +/- 1.0% alpha-actin(pos); beta-cat(Deltaex3-6): 49.9 +/- 2.4% alpha-actin(pos), P < 0.001). We conclude that beta-catenin depletion attenuates postinfarct LV remodeling in part through increased differentiation of GATA4(pos)/Sca-1(pos) resident cardiac progenitor cells.

Based on extensive preclinical data, glycogen synthase kinase-3 (GSK-3) has been proposed to be a viable drug target for a wide variety of disease states, ranging from diabetes to bipolar disorder. Since these new drugs, which will be more powerful GSK-3 inhibitors than lithium, may potentially be given to women of childbearing potential, and since it has controversially been suggested that lithium therapy might be linked to congenital cardiac defects, we asked whether GSK-3 family members are required for normal heart development in mice. We report that terminal cardiomyocyte differentiation was substantially blunted in Gsk3b(-/-) embryoid bodies. While GSK-3alpha-deficient mice were born without a cardiac phenotype, no live-born Gsk3b(-/-) pups were recovered. The Gsk3b(-/-) embryos had a double outlet RV, ventricular septal defects, and hypertrophic myopathy, with near obliteration of the ventricular cavities. The hypertrophic myopathy was caused by cardiomyocyte hyperproliferation without hypertrophy and was associated with increased expression and nuclear localization of three regulators of proliferation - GATA4, cyclin D1, and c-Myc. These studies, which we believe are the first in mammals to examine the role of GSK-3alpha and GSK-3beta in the heart using loss-of-function approaches, implicate GSK-3beta as a central regulator of embryonic cardiomyocyte proliferation and differentiation, as well as of outflow tract development. Although controversy over the teratogenic effects of lithium remains, our studies suggest that caution should be exercised in the use of newer, more potent drugs targeting GSK-3 in women of childbearing age.

Cell-transplantation therapy is a promising treatment option that is being actively explored as a way to repair cardiac muscle. The ultimate goal is to reconstitute the architecture of the cardiac muscle and to reestablish electrical propagation, while avoiding hypertrophy and scar formation. In this review, we focus on recent advances in the field as well as the difficulties encountered when the engraftment of cells into the host tissue is to be confirmed and functionally characterized. This is critical since incomplete or partial engraftment of transplanted cells within the host cardiac network exacerbates the heterogeneity already present in the injured myocardium and increases its propensity to arrhythmia. We conclude with a brief discussion of how the modulation of cell adhesion via modification of coupling proteins within transplanted cells may facilitate engraftment and alleviate the arrhythmogenic potential of cardiac grafts.

The heart is the first organ to function during vertebrate development and cardiac progenitors are among the first cell lineages to be established. In the chick, cardiac progenitors have been mapped in the epiblast of pre-streak embryos, and in the early gastrula they are located in the mid-primitive streak, from which they enter the mesoderm bilaterally. Signals controlling the specification of cardiac cells have been well documented; however, migration routes of cardiac progenitors have not been directly observed within the embryo and the factor(s) controlling their movement are not known. In addition, it is not clear how cell movement is coordinated with cell specification in the early embryo. Here we use live imaging to show that cardiac progenitors migrate in highly directed trajectories, which can be controlled by Wnt3a. Ectopic Wnt3a altered movement trajectories and caused cardia bifida. This was rescued by electroporation of dominant-negative DN-Wnt3a into prospective cardiac cells. Explant essays and mutant analysis showed that cellular guidance involved repulsion in response to Wnt3a and required RhoA function. It has been shown that Wnt3a inhibits cardiogenic cell specification through a beta-catenin-dependent pathway. On the basis of our results, we propose that Wnt3a concomitantly guides the movement of cardiac progenitors by a novel mechanism involving RhoA-dependent chemorepulsion.

Coronary artery disease is the most common cause of cardiac failure in the Western world, and to date there is no alternative to bypass surgery for severe coronary atherosclerosis. We report that c-kit-positive cardiac progenitor cells (CPCs) activated with insulin-like growth factor 1 and hepatocyte growth factor before their injection in proximity of the site of occlusion of the left coronary artery in rats, engrafted within the host myocardium forming temporary niches. Subsequently, CPCs divided and differentiated into endothelial cells and smooth muscle cells and, to a lesser extent, into cardiomyocytes. The acquisition of vascular lineages appeared to be mediated by the up-regulation of hypoxia-inducible factor 1alpha, which promoted the synthesis and secretion of stromal-derived factor 1 from hypoxic coronary vessels. Stromal-derived factor 1 was critical in the conversion of CPCs to the vascular fate. CPCs formed conductive and intermediate-sized coronary arteries together with resistance arterioles and capillaries. The new vessels were connected with the primary coronary circulation, and this increase in vascularization more than doubled myocardial blood flow in the infarcted myocardium. This beneficial effect, together with myocardial regeneration attenuated postinfarction dilated myopathy, reduced infarct size and improved function. In conclusion, locally delivered activated CPCs generate de novo coronary vasculature and may be implemented clinically for restoration of blood supply to the ischemic myocardium.