Postmitotic Cardiomyocytes Research Articles

Adult c-kitpos Cardiac Stem Cells Are Necessary and Sufficient for Functional Cardiac Regeneration and Repair

The epidemic of heart failure has stimulated interest in understanding cardiac regeneration. Evidence has been reported supporting regeneration via transplantation of multiple cell types, as well as replication of postmitotic cardiomyocytes. In addition, the adult myocardium harbors endogenous c-kit(pos) cardiac stem cells (eCSCs), whose relevance for regeneration is controversial. Here, using different rodent models of diffuse myocardial damage causing acute heart failure, we show that eCSCs restore cardiac function by regenerating lost cardiomyocytes. Ablation of the eCSC abolishes regeneration and functional recovery. The regenerative process is completely restored by replacing the ablated eCSCs with the progeny of one eCSC. eCSCs recovered from the host and recloned retain their regenerative potential in vivo and in vitro. After regeneration, selective suicide of these exogenous CSCs and their progeny abolishes regeneration, severely impairing ventricular performance. These data show that c-kit(pos) eCSCs are necessary and sufficient for the regeneration and repair of myocardial damage.

Early effects of acute doxorubicin treatment include increased apical caspase expression and activation in liver, but not heart, of fasted animals

Doxorubicin (DOX) is an effective chemotherapeutic agent, but known to cause cardiotoxicitv via induction of oxidative stress and apoptosis. DOX also causes hepatic toxicity, although mechanisms have not been as extensively described. Currently, the mechanism of DOX induced hepatic apoptosis is unknown. Moreover, despite the anorexic effects of DOX very few studies have controlled for this and, therefore, it is not clear whether changes in apoptosis markers and signaling in vivo are due to direct or indirect effects of DOX. Thus, we aimed to determine the early effects of DOX on expression and activation of apical caspases in heart and liver and to delineate these effects from mal consumption of food and water. Male F344 rats were injected IP with 20 mg/kg of DOX or saline. Once treated, all animals were fasted with no food or water until sacrifice 24 hours later. DOX did not affect expression or activation of caspase‐1, ‐8, ‐9, ‐12 in the heart at 24‐hour post treatment. However, DOX increased procaspase‐9 expression and caspase‐12 activation in liver. These results may suggest that at an early time point after acute DOX administration, the liver seems more vulnerable to DOX‐induced apoptosis compared to the heart. This may be explained by higher concentrations of DOX in liver compared to heart and/or the increased resistance to apoptosis in post‐mitotic cardiomyocytes compared to mitotic hepatocytes.

Anti-apoptotic function of the E2F transcription factor 4 (E2F4)/p130, a member of retinoblastoma gene family in cardiac myocytes

The E2F4–p130 transcriptional repressor complex is a cell-cycle inhibitor in mitotic cells. However, the role of E2F4/p130 in differentiated cells is largely unknown. We investigated the role of E2F4/p130 in the regulation of apoptosis in postmitotic cardiomyocytes. Here we demonstrate that E2F4 can inhibit hypoxia-induced cell death in isolated ventricular cardiomyocytes. As analyzed by chromatin immunoprecipitation, the E2F4–p130-repressor directly blocks transcription of essential apoptosis-related genes, E2F1, Apaf-1, and p73α through recruitment of histone deacetylase 1 (HDAC1). In contrast, diminution of the E2F4–p130–HDAC1-repressor and recruitment of E2F1 and histone acetylase activity to these E2F-regulated promoters is required for the execution of cell death. Expression of kinase-dead HDAC1.H141A or HDAC-binding deficient p130ΔHDAC1 abolishes the antiapoptotic effect of E2F4. Moreover, histological examination of E2F4−/− hearts revealed a markedly enhanced degree of cardiomyocyte apoptosis. Taken together, our genetic and biochemical data delineate an essential negative function of E2F4 in cardiac myocyte apoptosis.

Proteome wide determination of absolute turnover rates in mice

The administration of labeled amino acids has become the preferred method to measure the dynamics of protein turnover in living animals. Stable isotopes have been used as tracers to explore the amino acid flux in different metabolic pathways. Recently, the stable isotope labeling of amino acids in cell culture (SILAC), has been used to monitor individual protein turnover rates in cell culture and intact animals. This method is based on the incorporation of labeled essential amino acids resulting in appearance of labeled end products that can be quantified by mass spectrometry. We have performed an pulsed SILAC labeling of mice to calculate the protein turnover via the incorporation rate of labeled amino acid in post mitotic cardiomyocytes and could easily be applied to tissue from Lung.

Abstract P096: Eya4, a Transcription Cofactor Crucial in Acquired Heart Disease

Introduction: We previously identified a mutation in the human transcriptional cofactor Eya4 (E193) as cause of familial dilated cardiomyopathy (DCM) and heart failure. Upon interaction with real transcription factors such as Six family members, Eya4 is recruited to and interacts with target genes. One of the few known Eya-Six targets expressed in the heart is the cyclin-dependent kinase inhibitor p27kip1, which has been shown to inhibit hypertrophic growth in adult cardiomyocytes, is. We therefore hypothesize that Eya4/Six1 regulates targets relevant in normal cardiac function. Methods and results: We examined p27 expression in response to Eya4 in permanent mammalian cell lines. Western blot analysis demonstrated that an overexpression of Eya4 led to a significant downregulation of p27, whereas the overexpression of E193 had no effect on p27 levels. Studies using a p27 promoter fragment including Six1 consensus sites cloned in front of a Luciferase reporter gene proved Eya4 acts as a suppressor of p27 already at the transcriptional level, whereas E193 could not sufficiently inhibit p27 expression. Further transfection and knockdown experiments revealed that under basal conditions an Eya4 overexpression decreased protein synthesis in primary cardiac myocytes. We constructed transgenic mouse models with a constitutive myocardial overexpression of E193 and Eya4 to study the precise role of Eya4 in the heart. First analysis of these animals using magnetic resonance imaging to visualize cardiac structures in detail show that an overexpression of E193 leads to an age related onset of DCM as seen in an increase in LVEDV, whereas Eya4 overexpressing mice show no signs of heart disease. Conclusion: In summary, we identified a mutation in Eya4 to cause DCM. We now provide evidence that the Eya4/Six1 signalling cascade is also relevant in more common forms of acquired heart disease. Eya4/Six1 seems to regulate the expression of p27kip1, an important inhibitor of the development of hypertrophy in postmitotic cardiomyocytes. Studies using E193 overexpressing mice support our hypothesis whereas tempering the Eya4/Six1 signalling cascade disturbs cardiac physiology.

Skeletal Muscle Differentiation Evokes Endogenous XIAP to Restrict the Apoptotic Pathway

Myotube apoptosis occurs normally during muscle development and aging but it can lead to destruction of skeletal muscle in neuromuscular diseases. Therefore, understanding how myotube apoptosis is regulated is important for developing novel strategies for treatment of muscle loss. We investigated the regulation of apoptosis in skeletal muscle and report a striking increase in resistance to apoptosis following differentiation. We find mitotic C2C12 cells (myoblast-like cells) are sensitive to cytosolic cytochrome c microinjection. However, differentiated C2C12 cells (myotube-like cells) and primary myotubes are markedly resistant. This resistance is due to endogenous X-linked inhibitor of apoptotic protein (XIAP). Importantly, the selective difference in the ability of XIAP to block myotube but not myoblast apoptosis is not due to a change in XIAP but rather a decrease in Apaf-1 expression. This decrease in Apaf-1 links XIAP to caspase activation and death. Our findings suggest that in order for myotubes to die, they may degrade XIAP, functionally inactivate XIAP or upregulate Apaf-1. Importantly, we identify a role for endogenous Smac in overcoming XIAP to allow myotube death. However, in postmitotic cardiomyocytes, where XIAP also restricts apoptosis, endogenous Smac was not capable of overcoming XIAP to cause death. These results show that as skeletal muscle differentiate, they become resistant to apoptosis because of the ability of XIAP to regulate caspase activation. The increased restriction of apoptosis in myotubes is presumably important to ensure the long term survival of these postmitotic cells as they play a vital role in the physiology of organisms.

Stem cell engineering for treatment of heart diseases: Potentials and challenges

Heart disorders are a major health concern worldwide responsible for millions of deaths every year. Among the many disorders of the heart, myocardial infarction, which can lead to the development of congestive heart failure, arrhythmias, or even death, has the most severe social and economic ramifications. Lack of sufficient available donor hearts for heart transplantation, the only currently viable treatment for heart failure other than medical management options (ACE inhibition, beta blockade, use of AICDs, etc.) that improve the survival of patients with heart failure emphasises the need for alternative therapies. One promising alternative replaces cardiac muscle damaged by myocardial infarction with new contractile cardiomyocytes and vessels obtained through stem cell-based regeneration. We report on the state of the art of recovery of cardiac functions by using stem cell engineering. Current research focuses on (a) inducing stem cells into becoming cardiac cells before or after injection into a host, (b) growing replacement heart tissue in vitro, and (c) stimulating the proliferation of the post-mitotic cardiomyocytes in situ. The most promising treatment option for patients is the engineering of new heart tissue that can be implanted into damaged areas. Engineering of cardiac tissue currently employs the use of co-culture of stem cells with scaffold microenvironments engineered to improve tissue survival and enhance differentiation. Growth of heart tissue in vitro using scaffolds, soluble collagen, and cell sheets has unique advantages. To compensate for the loss of ventricular mass and contractility of the injured cardiomyocytes, different stem cell populations have been extensively studied as potential sources of new cells to ameliorate the injured myocardium and eventually restore cardiac function. Unresolved issues including insufficient cell generation survival, growth, and differentiation have led to mixed results in preclinical and clinical studies. Addressing these limitations should ensure the successful production of replacement heart tissue to benefit cardiac patients.

Notch activates cell cycle reentry and progression in quiescent cardiomyocytes

The inability of heart muscle to regenerate by replication of existing cardiomyocytes has engendered considerable interest in identifying developmental or other stimuli capable of sustaining the proliferative capacity of immature cardiomyocytes or stimulating division of postmitotic cardiomyocytes. Here, we demonstrate that reactivation of Notch signaling causes embryonic stem cell–derived and neonatal ventricular cardiomyocytes to enter the cell cycle. The proliferative response of neonatal ventricular cardiomyocytes declines as they mature, such that late activation of Notch triggers the DNA damage checkpoint and G2/M interphase arrest. Notch induces recombination signal-binding protein 1 for Jκ (RBP-Jκ)-dependent expression of cyclin D1 but, unlike other inducers, also shifts its subcellular distribution from the cytosol to the nucleus. Nuclear localization of cyclin D1 is independent of RBP-Jκ. Thus, the influence of Notch on nucleocytoplasmic localization of cyclin D1 is an unanticipated property of the Notch intracellular domain that is likely to regulate the cell cycle in multiple contexts, including tumorigenesis as well as cardiogenesis.

Cardiomyocyte proliferation in rat fetuses under normal conditions and after heart injury

Heart injury was inflicted to outbred albino rat fetuses on day 16 of prenatal development and cardiomyocyte proliferation was evaluated in the left ventricular zones adjacent to the wound and distant from it. The mitotic index of experimental animals was higher than in intact controls only for zones distant from the injury on day 10 after the intervention. The percentage of pathological metaphases gradually increased in all experimental groups, which causes doubt in the viability of postmitotic cardiomyocytes.

Suppression of Cardiomyocyte Hypertrophy by Inhibition of the Ubiquitin-Proteasome System

Inhibitors of the proteasome interfere with transcriptional regulation of growth signaling pathways and block cell cycle progression of mitotic cells. As growth signaling pathways are highly conserved between mitotic and postmitotic cells, we hypothesized that proteasome inhibition might also be a valuable approach to interfere with hypertrophic growth of postmitotic cardiomyocytes. To test this hypothesis, we analyzed the effects of proteasome inhibition on hypertrophic growth of neonatal rat cardiomyocytes. Partial inhibition of the proteasome effectively suppressed cardiomyocyte hypertrophy as determined by reduced cell size, inhibition of hypertrophy-mediated induction of RNA and protein synthesis, reduced expression of several hypertrophic marker genes, and diminished transcriptional activation of the BNP promotor. Importantly, suppression of hypertrophic growth was independent of the hypertrophic agonist used. Expressional profiling and subsequent Western blot and kinase assays revealed that proteasome inhibition induced a cellular stress response with reduced expression of conserved growth signaling mediators and impaired G1/S phase transition of cardiomyocytes. In hypertensive Dahl-salt sensitive rats, inhibition of the proteasome with low doses of the FDA approved proteasome inhibitor Velcade significantly reduced hypertrophic heart growth. Our data provide important insight into the suppressive effects of proteasome inhibitors on hypertrophic growth of cardiomyocytes and establish low-dose proteasome inhibition as a new and broad-spectrum approach to interfere with cardiac hypertrophy.

Critical Role for FoxO3a-Dependent Regulation of p21 CIP1/WAF1 in Response to Statin Signaling in Cardiac Myocytes

Statins are widely used clinical drugs that exert beneficial growth-suppressive effects in patients with cardiac hypertrophy. We investigated the role of the cell cycle inhibitor p21(CIP1/WAF1) (p21) in statin-dependent inhibition of hypertrophic growth in postmitotic cardiomyocytes. We demonstrate that lovastatin fails to inhibit cardiac hypertrophy to angiotensin II in p21(-/-) mice and that reconstitution of p21 function by TAT.p21 protein transduction can rescue statin action in these otherwise normally developed animals. Lovastatin specifically recruits the forkhead box FoxO3a transcription factor to the p21 promoter, mediating transcriptional transactivation of the p21 gene as analyzed in isolated primary cardiomyocytes. Lovastatin also stimulates protein kinase B/Akt kinase activity, and Akt-dependent phosphorylation forces p21 in the cytoplasm, where it inhibits Rho-kinases contributing to the suppression of cardiomyocyte hypertrophy. Loss of p21 or FoxO3a by RNA interference causes a general inhibition of lovastatin signal transduction. These results suggest that p21 functions as FoxO3a downstream target to mediate an statin-derived anti-hypertrophic response. Taken together, our genetic and biochemical data delineate an essential function of p21 for statin-dependent inhibition of cardiac myocyte hypertrophy.

Switch from Caspase-dependent to Caspase-independent Death during Heart Development: ESSENTIAL ROLE OF ENDONUCLEASE G IN ISCHEMIA-INDUCED DNA PROCESSING OF DIFFERENTIATED CARDIOMYOCYTES

Differentiated cardiomyocytes are resistant to caspase-dependent cell death; however, the mechanisms involved are still uncertain. We previously reported that low Apaf1 expression partially accounts for cardiomyocyte resistance to apoptosis. Here, we extend the knowledge on the molecular basis of cardiac resistance to caspase activation by showing that the whole caspase-dependent pathway is silenced during heart development. Experimental ischemia triggers caspase activation in embryonic cardiomyocytes and proliferating fibroblasts, but not in neonatal and adult cardiomyocytes. Ischemia induces the release of the proapoptotic factors cytochrome c, truncated-AIF, and EndoG from mitochondria in postnatal cardiomyocytes in the absence of caspase activation. On the one hand, lentiviral-driven knockdown of EndoG shows that this gene is essential for ischemia-induced DNA degradation in neonatal cardiomyocytes, but not in proliferating fibroblasts; on the other hand, the AIF gene is essential for high molecular DNA cleavage in fibroblasts, but not in postmitotic cardiomyocytes, where it plays a prosurvival role during reoxygenation. These results show the switch from caspase-dependent to caspase-independent death pathways after cardiac cell differentiation, and disclose the relevance of EndoG in the caspase-independent DNA processing of differentiated cardiomyocytes.

Adenoviral delivery of human CDC5 promotes G2/M progression and cell division in neonatal ventricular cardiomyocytes

Heart failure results from the cumulative death of cardiomyocytes, and the inability of remaining cells to regenerate. Efforts toward transcriptional reprogramming of cardiomyocytes by overexpressing E1A or E2F1 have been limited by the inability of cardiomyocytes to enter and complete mitosis. Human CDC5 (hCDC5), a component of the pre-mRNA splicing complex, has been shown to regulate G2/M transit in asynchronously dividing cells. We now show that co-infection of recombinant adenoviruses expressing E1A/E1B and hCDC5 promotes cell cycle re-entry and G2/M progression in post-mitotic cardiomyocytes. Co-expression of E1A/E1B and hCDC5 induced nuclear localization of cyclin-dependent kinase 1 and cyclin B1, and was sufficient to promote mitotic entry as determined by an increase in mitotic index only in co-infected cells. E1A/E1B and hCDC5 promoted cell division, as evidenced by an increase in the number of cardiomyocytes following co-infection. Thus, overexpression of E1A/E1B and hCDC5 resulted in cell cycle re-entry, DNA synthesis, cell division, and an increase in cardiomyocyte number, suggesting the formation of new cardiomyocytes. These studies suggest that G1/S-phase transcriptional regulators, in combination with pre-mRNA splicing factors, such as CDC5, that regulate rate-limiting G2/M target genes may prove useful in developing therapies to stimulate myocardial regeneration.

Thyroid hormone induces cyclin D1 nuclear translocation and DNA synthesis in adult rat cardiomyocytes

Although mammalian cardiomyocytes lose their proliferative capacity after birth, there is evidence that postmitotic cardiomyocytes can proliferate provided that cyclin D1 accumulates in the nucleus. Here we show by Northern blot, Western analysis, and immunohistochemistry that 3,5,3'-triiodothyronine (T3) treatment of adult rats caused an increase of cyclin D1 mRNA and protein levels. The increased cyclin D1 protein content was associated with its translocation into the nucleus of cardiomyocytes. These changes were accompanied by the re-entry of cardiomyocytes into the cell cycle, as demonstrated by increased levels of cyclin A, PCNA, and incorporation of bromodeoxyuridine into DNA (labeling index was 30.2% in T3-treated rats vs. 2.2% in controls). Entry into the S phase was associated with an increased mitotic activity as demonstrated by positivity of cardiomyocyte nuclei to antibodies anti-phosphohistone-3, a specific marker of the mitotic phase (mitotic index was 3.01/1000 cardiomyocte nuclei in hyperthyroid rats vs. 0.04 in controls). No biochemical or histological signs of tissue damage were observed in the heart of T3-treated rats. These results demonstrated that T3 treatment is associated with a re-entry of cardiomyocytes into the cell cycle and so may be important for the development of future therapeutic strategies aimed at inducing proliferation of cardiomyocytes.

Reduced Apaf-1 levels in cardiomyocytes engage strict regulation of apoptosis by endogenous XIAP

Overexpression studies have identified X-linked inhibitor of apoptosis protein (XIAP) as a potent inhibitor of caspases. However, the exact function of endogenous XIAP in regulating mammalian apoptosis is less clear. Endogenous XIAP strictly regulates cytochrome c–dependent caspase activation in sympathetic neurons but not in many mitotic cells. We report that postmitotic cardiomyocytes, unlike fibroblasts, are remarkably resistant to cytosolic microinjection of cytochrome c. The cardiomyocyte resistance to cytochrome c is mediated by endogenous XIAP, as XIAP-deficient cardiomyocytes die rapidly with cytosolic cytochrome c alone. Importantly, we found that cardiomyocytes, like neurons, have markedly reduced Apaf-1 levels and that this decrease in Apaf-1 is directly linked to the tight regulation of caspase activation by XIAP. These data identify an important function of XIAP in cardiomyocytes and point to a striking similarity in the regulation of apoptosis in postmitotic cells.

Endomitosis and polyploidization of myocardial cells in the periphery of human acute myocardial infarction

Although the genetic program for reinitiating DNA synthesis exists in post-mitotic cardiomyocytes, and it was reported that in human acute myocardial infarction (AMI) a significant proportion of myocytes enter mitosis, the rule is that the lost tissue is replaced by a collagen scar. The purpose of this study was to search for the basis of this discordance in order to devise future strategies to induce division of myocytes into daughter cells that may replace the lost tissue with contractile cells. In 15 human hearts with 1- to 21-day-old infarcts, the expression of the cell cycle proteins Ki67 antigen, cyclins D, A, and B1, the presence of mitotic bodies, and the ploidy status were investigated with immunoenzymatic methods, light and laser confocal microscopy, and densitometry in the myocytes surrounding the infarct area. In 7- to 13-day-old infarcts, 11.61+/-6.94% of the myocytes presented Ki67+ nuclei, and a lower proportion presented cyclins D, A, and B. At earlier and later times, the proportion of Ki67+ myocytes was significantly lower. Although under confocal microscopy and fluorescent labels, some of the Ki67+ myocytes appeared to be in different stages of mitosis, with Nomarski optics and hematoxylin counterstaining, the condensed chromosomes, although arranged in metaphase and anaphase plates or split in sister chromatids, were always located within a preserved nuclear envelope, indicating the presence of endomitosis. Conventional mitosis was exceptionally observed. In the 14- and 21-day-old infarcts, the ploidy of the myocytes adjacent to the infarct was significantly higher than in distant zones. These observations indicate that in human infarcts, entrance of cardiomyocytes into the cell cycle is transient and that endomitosis, leading to polyploidy, rather than mitosis, leading to karyokinesis, is the final fate of cycling cells. Both observations may account for the discordance between the regenerative ability of myocytes and the lack of an efficient reparative process in human AMI.

Reactivation of the Mitosis-Promoting Factor in Postmitotic Cardiomyocytes

Cardiomyocytes cease to divide shortly after birth and an irreversible cell cycle arrest is evident accompanied by the downregulation of cyclin-dependent kinase activities. To get a better understanding of the cardiac cell cycle and its regulation, the effect of functional recovery of the mitosis-promoting factor (MPF) consisting of cyclin B1 and the cyclin-dependent kinase Cdc2 was assessed in primary cultures of postmitotic ventricular adult rat cardiomyocytes (ARC). Gene transfer into ARC was achieved using the adenovirus-enhanced transferrinfection system that was characterized by the absence of cytotoxic events. Simultaneous ectopic expression of wild-type versions of cyclin B1 and Cdc2 was sufficient to induce MPF activity. Reestablished MPF resulted in a mitotic phenotype, marked by an abnormal condensation of the nuclei, histone H3 phosphorylation and variable degree of decay of the contractile apparatus. Although a complete cell division was not observed, the results provided conclusive evidence that cell cycle-related events in postmitotic cardiomyocytes could be triggered by genetic intervention downstream of the G1/S checkpoint. This will be of importance to design novel strategies to overcome the proliferation arrest in adult cardiomyocytes.

Critical role of cyclin D1 nuclear import in cardiomyocyte proliferation.

Mammalian cardiomyocytes irreversibly lose their capacity to proliferate soon after birth, yet the underlying mechanisms have been unclear. Cyclin D1 and its partner, cyclin-dependent kinase 4 (CDK4), are important for promoting the G1-to-S phase progression via phosphorylation of the retinoblastoma (Rb) protein. Mitogenic stimulation induces hypertrophic cell growth and upregulates expression of cyclin D1 in postmitotic cardiomyocytes. In the present study, we show that, in neonatal rat cardiomyocytes, D-type cyclins and CDK4 were predominantly cytoplasmic, whereas Rb remained in an underphosphorylated state. Ectopically expressed cyclin D1 localized in the nucleus of fetal but not neonatal cardiomyocytes. To target cyclin D1 to the nucleus efficiently, we constructed a variant of cyclin D1 (D1NLS), which directly linked to nuclear localization signals (NLSs). Coinfection of recombinant adenoviruses expressing D1NLS and CDK4 induced Rb phosphorylation and CDK2 kinase activity. Furthermore, D1NLS/CDK4 was sufficient to promote the reentry into the cell cycle, leading to cell division. The number of cardiomyocytes coinfected with these viruses increased 3-fold 5 days after infection. Finally, D1NLS/CDK4 promoted cell cycle reentry of cardiomyocytes in adult hearts injected with these viruses, evaluated by the expression of Ki-67, which is expressed in proliferating cells in all phases of the cell cycle, and BrdU incorporation. Thus, postmitotic cardiomyocytes have the potential to proliferate provided that cyclin D1/CDK4 accumulate in the nucleus, and the prevention of their nuclear import plays a critical role as a physical barrier to prevent cardiomyocyte proliferation. Our results provide new insights into the development of therapeutics strategies to induce regeneration of cardiomyocytes. The full text of this article is available at http://www.circresaha.org.

Stem cell differentiation requires a paracrine pathway in the heart.

Members of the transforming growth factor beta1 (TGF-beta) superfamily--namely, TGF-beta and BMP2--applied to undifferentiated murine embryonic stem cells up-regulated mRNA of mesodermal (Brachyury) and cardiac specific transcription factors (Nkx2.5, MEF2C). Embryoid bodies generated from stem cells primed with these growth factors demonstrated an increased potential for cardiac differentiation with a significant increase in beating areas and enhanced myofibrillogenesis. In an environment of postmitotic cardiomyocytes, stem cells engineered to express a fluorescent protein under the control of a cardiac promoter differentiated into fluorescent ventricular myocytes beating in synchrony with host cells, a process significantly enhanced by TGF-beta or BMP2. In vitro, disruption of the TGF-beta/BMP signaling pathways by latency-associated peptide and/or noggin prevented differentiation of stem cells. In fact, only host cells that secrete a TGF-beta family member induced a cardiac phenotype in stem cells. In vivo, transplantation of stem cells into heart also resulted in cardiac differentiation provided that TGF-beta/BMP2 signaling was intact. In infarcted myocardium, grafted stem cells differentiated into functional cardiomyocytes integrated with surrounding tissue, improving contractile performance. Thus, embryonic stem cells are directed to differentiate into cardiomyocytes by signaling mediated through TGF-beta/BMP2, a cardiac paracrine pathway required for therapeutic benefit of stem cell transplantation in diseased heart.

The role of stem cells in skeletal and cardiac muscle repair.

In postnatal muscle, skeletal muscle precursors (myoblasts) can be derived from satellite cells (reserve cells located on the surface of mature myofibers) or from cells lying beyond the myofiber, e.g., interstitial connective tissue or bone marrow. Both of these classes of cells may have stem cell properties. In addition, the heretical idea that post-mitotic myonuclei lying within mature myofibers might be able to re-form myoblasts or stem cells is examined and related to recent observations for similar post-mitotic cardiomyocytes. In adult hearts (which previously were not considered capable of repair), the role of replicating endogenous cardiomyocytes and the recruitment of other (stem) cells into cardiomyocytes for new cardiac muscle formation has recently attracted much attention. The relative contribution of these various sources of precursor cells in postnatal muscles and the factors that may enhance stem cell participation in the formation of new skeletal and cardiac muscle in vivo are the focus of this review. We concluded that, although many endogenous cell types can be converted to skeletal muscle, the contribution of non-myogenic cells to the formation of new postnatal skeletal muscle in vivo appears to be negligible. Whether the recruitment of such cells to the myogenic lineage can be significantly enhanced by specific inducers and the appropriate microenvironment is a current topic of intense interest. However, dermal fibroblasts appear promising as a realistic alternative source of exogenous myoblasts for transplantation purposes. For heart muscle, experiments showing the participation of bone marrow-derived stem cells and endothelial cells in the repair of damaged cardiac muscle are encouraging.