De novo cardiomyocytes from within the activated adult heart after injury

A significant bottleneck in cardiovascular regenerative medicine is the identification of a viable source of stem/progenitor cells that could contribute new muscle after ischaemic heart disease and acute myocardial infarction. A therapeutic ideal--relative to cell transplantation--would be to stimulate a resident source, thus avoiding the caveats of limited graft survival, restricted homing to the site of injury and host immune rejection. Here we demonstrate in mice that the adult heart contains a resident stem or progenitor cell population, which has the potential to contribute bona fide terminally differentiated cardiomyocytes after myocardial infarction. We reveal a novel genetic label of the activated adult progenitors via re-expression of a key embryonic epicardial gene, Wilm's tumour 1 (Wt1), through priming by thymosin β4, a peptide previously shown to restore vascular potential to adult epicardium-derived progenitor cells with injury. Cumulative evidence indicates an epicardial origin of the progenitor population, and embryonic reprogramming results in the mobilization of this population and concomitant differentiation to give rise to de novo cardiomyocytes. Cell transplantation confirmed a progenitor source and chromosome painting of labelled donor cells revealed transdifferentiation to a myocyte fate in the absence of cell fusion. Derived cardiomyocytes are shown here to structurally and functionally integrate with resident muscle; as such, stimulation of this adult progenitor pool represents a significant step towards resident-cell-based therapy in human ischaemic heart disease.     

In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes

The reprogramming of adult cells into pluripotent cells or directly into alternative adult cell types holds great promise for regenerative medicine. We reported previously that cardiac fibroblasts,which represent 50%of the cells in the mammalian heart, can be directly reprogrammed to adult cardiomyocyte-like cells in vitro by the addition of Gata4, Mef2c and Tbx5 (GMT). Here we use genetic lineage tracing to show that resident non-myocytes in the murine heart can be reprogrammed into cardiomyocyte-like cells in vivo by local delivery of GMT after coronary ligation. Induced cardiomyocytes became binucleate, assembled sarcomeres and had cardiomyocyte-like gene expression. Analysis of single cells revealed ventricular cardiomyocyte-like action potentials, beating upon electrical stimulation, and evidence of electrical coupling. In vivo delivery of GMT decreased infarct size and modestly attenuated cardiac dysfunction up to 3 months after coronary ligation. Delivery of the pro-angiogenic and fibroblast-activating peptide, thymosin b4, along with GMT, resulted in further improvements in scar area and cardiac function. These findings demonstrate that cardiac fibroblasts can be reprogrammed into cardiomyocyte-like cells in their native environment for potential regenerative purposes.     

Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts

The mammalian heart has a very limited regenerative capacity and, hence, heals by scar formation. Recent reports suggest that haematopoietic stem cells can transdifferentiate into unexpected phenotypes such as skeletal muscle, hepatocytes, epithelial cells, neurons, endothelial cells and cardiomyocytes, in response to tissue injury or placement in a new environment. Furthermore, transplanted human hearts contain myocytes derived from extra-cardiac progenitor cells, which may have originated from bone marrow. Although most studies suggest that transdifferentiation is extremely rare under physiological conditions, extensive regeneration of myocardial infarcts was reported recently after direct stem cell injection, prompting several clinical trials. Here, we used both cardiomyocyte-restricted and ubiquitously expressed reporter transgenes to track the fate of haematopoietic stem cells after 145 transplants into normal and injured adult mouse hearts. No transdifferentiation into cardiomyocytes was detectable when using these genetic techniques to follow cell fate, and stem-cell-engrafted hearts showed no overt increase in cardiomyocytes compared to sham-engrafted hearts. These results indicate that haematopoietic stem cells do not readily acquire a cardiac phenotype, and raise a cautionary note for clinical studies of infarct repair.     

Insulin improves cardiac myocytes contractile function recovery in simulated ischemia-reperfusion： Key role of Akt

There are abundant insulin receptors on the membrane of myocytes, which renders cardiomyocytes typical target cells of insulin. Accumulating evidence has indicated that abnormal insulin level is an important predisposing factor in diabetes-related cardiovascular disorders and may contribute to the development of cardiovascular diseases, such as coronary heart diseaseand heart failure. It has been shown that insulin couldreduce infarct size in experimental animals subjected to myocardial ischem…     

Thymosin beta4 induces adult epicardial progenitor mobilization and neovascularization

Cardiac failure has a principal underlying aetiology of ischaemic damage arising from vascular insufficiency. Molecules that regulate collateral growth in the ischaemic heart also regulate coronary vasculature formation during embryogenesis. Here we identify thymosin beta4 (Tbeta4) as essential for all aspects of coronary vessel development in mice, and demonstrate that Tbeta4 stimulates significant outgrowth from quiescent adult epicardial explants, restoring pluripotency and triggering differentiation of fibroblasts, smooth muscle cells and endothelial cells. Tbeta4 knockdown in the heart is accompanied by significant reduction in the pro-angiogenic cleavage product N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP). Although injection of AcSDKP was unable to rescue Tbeta4 mutant hearts, it significantly enhanced endothelial cell differentiation from adult epicardially derived precursor cells. This study identifies Tbeta4 and AcSDKP as potent stimulators of coronary vasculogenesis and angiogenesis, and reveals Tbeta4-induced adult epicardial cells as a viable source of vascular progenitors for continued renewal of regressed vessels at low basal level or sustained neovascularization following cardiac injury.     

Akt–Oct4 regulatory circuit in pluripotent stem cells

Oct4 is mainly expressed in embryonic stem cells(ESCs),germline stem cells,and embryonal carcinoma cells(ECCs)and plays an indispensable role in maintaining the pluripotency and self-renewal of these pluripotent stem cells.Akt serine/threonine kinase,a wellestablished anti-apoptosis and cell survival factor,has also been implicated as an important regulator of stemness.Emerging evidence indicated that Oct4 is reciprocally connected to Akt via a number of routes,and moreover,a direct interaction between Oct4 and Akt has recently been revealed.These components collectively form the Akt–Oct4 regulatory circuit.In this review,we summarize our current knowledge about the Akt–Oct4 regulatory circuit in ESCs and discuss its alterations in ECCs that may underlie the tumorigenesis of pluripotent stem cells.     

Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population

The functional heart is comprised of distinct mesoderm-derived lineages including cardiomyocytes, endothelial cells and vascular smooth muscle cells. Studies in the mouse embryo and the mouse embryonic stem cell differentiation model have provided evidence indicating that these three lineages develop from a common Flk-1(+) (kinase insert domain protein receptor, also known as Kdr) cardiovascular progenitor that represents one of the earliest stages in mesoderm specification to the cardiovascular lineages. To determine whether a comparable progenitor is present during human cardiogenesis, we analysed the development of the cardiovascular lineages in human embryonic stem cell differentiation cultures. Here we show that after induction with combinations of activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF, also known as FGF2), vascular endothelial growth factor (VEGF, also known as VEGFA) and dickkopf homolog 1 (DKK1) in serum-free media, human embryonic-stem-cell-derived embryoid bodies generate a KDR(low)/C-KIT(CD117)(neg) population that displays cardiac, endothelial and vascular smooth muscle potential in vitro and, after transplantation, in vivo. When plated in monolayer cultures, these KDR(low)/C-KIT(neg) cells differentiate to generate populations consisting of greater than 50% contracting cardiomyocytes. Populations derived from the KDR(low)/C-KIT(neg) fraction give rise to colonies that contain all three lineages when plated in methylcellulose cultures. Results from limiting dilution studies and cell-mixing experiments support the interpretation that these colonies are clones, indicating that they develop from a cardiovascular colony-forming cell. Together, these findings identify a human cardiovascular progenitor that defines one of the earliest stages of human cardiac development.     

Overexpressing dominant negative MyD88 induces cardiac dysfunction in transgenic mice

Myeloid differentiation protein-88 (MyD88) is a crucial adaptor protein in the innate immune response. A protective role for MyD88 in normal cardiac function has been proposed in a surgical hypertrophic model. To assess the in vivo role of MyD88 in cardiac remodeling, we generated transgenic mice with cardiac-restricted expression of a dominant negative mutant of MyD88 (dnMyD88). Surprisingly, dnMyD88 transgenic mice displayed characteristic features of heart failure; including heart weight increase, cardiomyocytes enlargement, interstitial fibrosis, and re-expression of “fetal” genes. Echocardiographic examination of dnMyD88 hearts revealed dilated chamber volume and reduced cardiac contractility. DnMyD88 mice died from heart failure before they were 7 months old, as shown by Kaplan-Meier analysis. Additionally, the heart failure phenotype of dnMyD88 mice was associated with abnormal activation of the Akt/GSK-3β signaling pathway. These data provide the first evidence that normal MyD88 signaling is crucial for maintaining the physiological function of the adult heart.     

干细胞与心肌再生

由于成年心肌细胞通常不能再生,严重的心肌损伤会导致心肌不可逆的重构坏死, 从而发生心功能失调. 干细胞再生治疗为心肌再生提供了很好的策略. 为了寻找合适的干细胞类型, 促进心肌再生, 有效改善心功能, 需要更好地了解心肌修复和再生的分子基础. 已有研究发现多种干细胞可促进心肌再生. 描述了骨髓干细胞的促血管新生及心肌分化的能力在心梗治疗中的作用, 还讨论了心脏侧群干细胞以及诱导型多能干细胞在心肌再生中的作用和分子机制. 所阐述的最新数据有利于拓展干细胞治疗的有效潜能及临床影响.     

A genetic blueprint for cardiac development

Congenital heart disease is the leading non-infectious cause of death in children. It is becoming increasingly clear that many cardiac abnormalities once thought to have multifactorial aetiologies are attributable to mutations in developmental control genes. The consequences of these mutations can be manifest at birth as life-threatening cardiac malformations or later as more subtle cardiac abnormalities. Understanding the genetic underpinnings of cardiac development has important implications not only for understanding congenital heart disease, but also for the possibility of cardiac repair through genetic reprogramming of non-cardiac cells to a cardiogenic fate.     

Essential roles of PI(3)K-p110beta in cell growth, metabolism and tumorigenesis

On activation by receptors, the ubiquitously expressed class IA isoforms (p110alpha and p110beta) of phosphatidylinositol-3-OH kinase (PI(3)K) generate lipid second messengers, which initiate multiple signal transduction cascades. Recent studies have demonstrated specific functions for p110alpha in growth factor and insulin signalling. To probe for distinct functions of p110beta, we constructed conditional knockout mice. Here we show that ablation of p110beta in the livers of the resulting mice leads to impaired insulin sensitivity and glucose homeostasis, while having little effect on phosphorylation of Akt, suggesting the involvement of a kinase-independent role of p110beta in insulin metabolic action. Using established mouse embryonic fibroblasts, we found that removal of p110beta also had little effect on Akt phosphorylation in response to stimulation by insulin and epidermal growth factor, but resulted in retarded cell proliferation. Reconstitution of p110beta-null cells with a wild-type or kinase-dead allele of p110beta demonstrated that p110beta possesses kinase-independent functions in regulating cell proliferation and trafficking. However, the kinase activity of p110beta was required for G-protein-coupled receptor signalling triggered by lysophosphatidic acid and had a function in oncogenic transformation. Most strikingly, in an animal model of prostate tumour formation induced by Pten loss, ablation of p110beta (also known as Pik3cb), but not that of p110alpha (also known as Pik3ca), impeded tumorigenesis with a concomitant diminution of Akt phosphorylation. Taken together, our findings demonstrate both kinase-dependent and kinase-independent functions for p110beta, and strongly indicate the kinase-dependent functions of p110beta as a promising target in cancer therapy.     

Chloride conductance regulated by cyclic AMP-dependent protein kinase in cardiac myocytes

In heart cells, cyclic AMP-dependent protein kinase (PKA) regulates calcium- and potassium-ion current by phosphorylating the ion channels or closely associated regulatory proteins. We report here that isoprenaline induced large chloride-ion currents in voltage-clamped, internally-dialysed myocytes from guinea-pig ventricles. The Cl- current could be activated by intracellular dialysis with cAMP or the catalytic subunit of PKA, indicating regulation by phosphorylation. In approximately symmetrical solutions of high Cl- concentration, the macroscopic cardiac Cl- current showed little rectification, unlike the single-channel current in PKA-regulated Cl- channels of airway epithelial cells. But, like epithelial Cl- -channel currents, the cardiac Cl- current was sensitive to the distilbene,4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS). In the absence of kinase activation, cardiac sarcolemmal Cl- conductance was negligible. During beta-adrenergic stimulation of the heart, this novel Cl- conductance should accelerate action-potential repolarization and so protect impulse propagation in the face of the possibly arrhythmogenic increases in heart rate and in calcium entry into the cells.     

Stem-cell-based therapy and lessons from the heart

The potential usefulness of human embryonic stem cells for therapy derives from their ability to form any cell in the body. This potential has been used to justify intensive research despite some ethical concerns. In parallel, scientists have searched for adult stem cells that can be used as an alternative to embryonic cells, and, for the heart at least, these efforts have led to promising results. However, most adult cardiomyocytes are unable to divide and form new cardiomyocytes and would therefore be unable to replace those lost as a result of disease. Basic questions--for example, whether cardiomyocyte replacement or alternatives, such as providing the damaged heart with new blood vessels or growth factors to activate resident stem cells, are the best approach--remain to be fully addressed. Despite this, preclinical studies on cardiomyocyte transplantation in animals and the first clinical trials with adult stem cells have recently been published with mixed results.     

Binding of paxillin to alpha4 integrins modifies integrin-dependent biological responses

The alpha4 integrins are indispensable for embryogenesis, haematopoiesis and immune responses, possibly because alpha4 regulates cellular functions differently from other integrins through its cytoplasmic tail. We used novel mimics of the alpha4 tail to identify molecules that could account for alpha4-specific signalling. Here we report that the alpha4 tail, but not several other alpha-subunit tails, binds tightly to the signalling adaptor paxillin. Paxillin physically associated with alpha4 integrins in Jurkat T cells at high stoichiometry, and joining the alpha4 tail to alphaIIb resulted in a complex of integrin alphaIIbbeta3 with paxillin. This association markedly enhanced the rates of alphaIIbbeta3-dependent phosphorylation of focal adhesion kinase and cell migration. It also reduced cell spreading, focal adhesion and stress fibre formation. A point mutation within the alpha4 tail that disrupts paxillin binding reversed all of these effects. Furthermore, alpha4beta1-dependent adhesion to VCAM-1 led to spreading of mouse embryonic fibroblasts derived from paxillin-null but not from wild-type mice. Thus, the tight association of paxillin with the alpha4 tail leads to distinct biochemical and biological responses to integrin-mediated cell adhesion.     

Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts

Transplantation studies in mice and rats have shown that human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) can improve the function of infarcted hearts, but two critical issues related to their electrophysiological behaviour in vivo remain unresolved. First, the risk of arrhythmias following hESC-CM transplantation in injured hearts has not been determined. Second, the electromechanical integration of hESC-CMs in injured hearts has not been demonstrated, so it is unclear whether these cells improve contractile function directly through addition of new force-generating units. Here we use a guinea-pig model to show that hESC-CM grafts in injured hearts protect against arrhythmias and can contract synchronously with host muscle. Injured hearts with hESC-CM grafts show improved mechanical function and a significantly reduced incidence of both spontaneous and induced ventricular tachycardia. To assess the activity of hESC-CM grafts in vivo, we transplanted hESC-CMs expressing the genetically encoded calcium sensor, GCaMP3 (refs 4, 5). By correlating the GCaMP3 fluorescent signal with the host ECG, we found that grafts in uninjured hearts have consistent 1:1 host–graft coupling. Grafts in injured hearts are more heterogeneous and typically include both coupled and uncoupled regions. Thus, human myocardial grafts meet physiological criteria for true heart regeneration, providing support for the continued development of hESC-based cardiac therapies for both mechanical and electrical repair.