Georgina M. Ellison

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.

MicroRNA-133 controls vascular smooth muscle cell phenotypic switch in vitro and vascular remodeling in vivo

Rationale: MicroRNA (miR)-1 and -133 play a crucial role in skeletal and cardiac muscle biology and pathophysiology. However, their expression and regulation in vascular cell physiology and disease is currently unknown. Objective: The aim of the present study was to evaluate the role, if any, of miR-1 and miR-133 in vascular smooth muscle cell (VSMC) phenotypic switch in vitro and in vivo. Methods and Results: We demonstrate here that miR-133 is robustly expressed in vascular smooth muscle cells (VSMCs) in vitro and in vivo, whereas miR-1 vascular levels are negligible. miR-133 has a potent inhibitory role on VSMC phenotypic switch in vitro and in vivo, whereas miR-1 does not have any relevant effect per se. miR-133 expression is regulated by extracellular signal–regulated kinase 1/2 activation and is inversely correlated with VSMC growth. Indeed, miR-133 decreases when VSMCs are primed to proliferate in vitro and following vascular injury in vivo, whereas it increases when VSMCs are coaxed back to quiescence in vitro and in vivo. miR-133 loss- and gain-of-function experiments show that miR-133 plays a mechanistic role in VSMC growth. Accordingly, adeno-miR-133 reduces but anti-miR-133 exacerbates VSMC proliferation and migration in vitro and in vivo. miR-133 specifically suppresses the transcription factor Sp-1 expression in vitro and in vivo and through Sp-1 repression regulates smooth muscle gene expression. Conclusions: Our data show that miR-133 is a key regulator of vascular smooth muscle cell phenotypic switch in vitro and in vivo, suggesting its potential therapeutic application for vascular diseases.

Endogenous cardiac stem cell activation by insulin-like growth factor-1/hepatocyte growth factor intracoronary injection fosters survival and regeneration of the infarcted pig heart.

OBJECTIVES The purpose of this study was to test the ability of insulin-like growth factor (IGF)-1/hepatocyte growth factor (HGF) to activate resident endogenous porcine cardiac stem/progenitor cells (epCSCs) and to promote myocardial repair through a clinically applicable intracoronary injection protocol in a pig model of myocardial infarction (MI) relevant to human disease. BACKGROUND In rodents, cardiac stem/progenitor cell (CSC) transplantation as well as in situ activation through intramyocardial injection of specific growth factors has been shown to result in myocardial regeneration after acute myocardial infarction (AMI). METHODS Acute MI was induced in pigs by a 60-min percutaneous transluminal coronary angiography left anterior descending artery occlusion. The IGF-1 and HGF were co-administered through the infarct-related artery in a single dose (ranging from 0.5 to 2 μg HGF and 2 to 8 μg IGF-1) 30 min after coronary reperfusion. Pigs were sacrificed 21 days later for dose-response relationship evaluation by immunohistopathology or 2 months later for cardiac function evaluation by cardiac magnetic resonance imaging. RESULTS The IGF-1/HGF activated c-kit positive-CD45 negative epCSCs and increased their myogenic differentiation in vitro. The IGF-1/HGF, in a dose-dependent manner, improved cardiomyocyte survival, and reduced fibrosis and cardiomyocyte reactive hypertrophy. It significantly increased c-kit positive-CD45 negative epCSC number and fostered the generation of new myocardium (myocytes and microvasculature) in infarcted and peri-infarct/border regions at 21 and 60 days after AMI. The IGF-1/HGF reduced infarct size and improved left ventricular function at 2 months after AMI. CONCLUSIONS In an animal model of AMI relevant to the human disease, intracoronary administration of IGF-1/HGF is a practical and effective strategy to reduce pathological cardiac remodeling, induce myocardial regeneration, and improve ventricular function.

Physiological cardiac remodelling in response to endurance exercise training: cellular and molecular mechanisms

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 Athletes 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.

Acute β-Adrenergic Overload Produces Myocyte Damage through Calcium Leakage from the Ryanodine Receptor 2 but Spares Cardiac Stem Cells

A hyperadrenergic state is a seminal aspect of chronic heart failure. Also, “Takotsubo stress cardiomyopathy,” is associated with increased plasma catecholamine levels. The mechanisms of myocyte damage secondary to excess catecholamine exposure as well as the consequence of this neurohumoral burst on cardiac stem cells (CSCs) are unknown. Cardiomyocytes and CSCs were exposed to high doses of isoproterenol (ISO), in vivo and in vitro. Male Wistar rats received a single injection of ISO (5 mg kg-1) and were sacrificed 1, 3, and 6 days later. In comparison with controls, LV function was impaired in rats 1 day after ISO and started to improve at 3 days. The fraction of dead myocytes peaked 1 day after ISO and decreased thereafter. ISO administration resulted in significant ryanodine receptor 2 (RyR2) hyperphosphorylation and RyR2-calstabin dissociation. JTV519, a RyR2 stabilizer, prevented the ISO-induced death of adult myocytes in vitro. In contrast, CSCs were resistant to the acute neurohumoral overload. Indeed, CSCs expressed a decreased and inverted complement of β1/β2-adrenoreceptors and absence of RyR2, which may explain their survival to ISO insult. Thus, a single injection of ISO causes diffuse myocyte death through Ca2+ leakage secondary to the acutely dysfunctional RyR2. CSCs are resistant to the noxious effects of an acute hyperadrenergic state and through their activation participate in the response to the ISO-induced myocardial injury. The latter could contribute to the ability of the myocardium to rapidly recover from acute hyperadrenergic damage.

Characterization of Long-Term Cultured c-kit+ Cardiac Stem Cells Derived From Adult Rat Hearts

Previous studies have revealed c-kit-positive (c-kit(+)) cardiac stem cells (CSCs) in the adult mammalian heart and these cells could be a suitable cell source for heart regeneration therapy. However, these cells have not been fully evaluated in terms of characterization and effect of long-term culture, which is necessary for their safe and optimal usage. Therefore, we isolated c-kit(+) CSCs from adult rat hearts to characterize these cells and investigate stability over long-term culture. We performed isolations of c-kit(+) CSCs 11 times and passaged them 40 times in a bulk culture system; we termed these cultures, bulk culture CSCs (CSC-BC). c-kit(+) CSCs expressed stemness genes and exhibited stem cell properties of single cell-derived clone formation, cardiosphere generation, and potential to differentiate into the three main cardiac lineages: cardiomyocyte, smooth muscle, and endothelial cells in vitro. Over long-term culture, some CSC-BC up-regulated GATA-4 expression, which resulted in enhanced cardiomyocyte differentiation, suggesting that the GATA-4 high c-kit(+) CSCs have potent cardiac regenerative potential. We also observed the spontaneous differentiation into cells other than cardiac lineages, such as adipocyte and skeletal myocyte. This effect of long-term culture on the c-kit(+) CSCs has not been previously reported. Interestingly, when c-kit(+) CSCs were co-cultured with adult rat cardiomyocytes, we found increased cardiomyocyte survival, and the growth factors, insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF), appeared to be responsible factors. The present study suggests that c-kit(+) CSCs have great therapeutic potential yet should be further investigated and optimized as a cell source for regenerative therapies prior to transplantation.

The adult heart responds to increased workload with physiologic hypertrophy, cardiac stem cell activation, and new myocyte formation

Aims It is a dogma of cardiovascular pathophysiology that the increased cardiac mass in response to increased workload is produced by the hypertrophy of the pre-existing myocytes. The role, if any, of adult-resident endogenous cardiac stem/progenitor cells (eCSCs) and new cardiomyocyte formation in physiological cardiac remodelling remains unexplored. Methods and results In response to regular, intensity-controlled exercise training, adult rats respond with hypertrophy of the pre-existing myocytes. In addition, a significant number (∼7%) of smaller newly formed BrdU-positive cardiomyocytes are produced by the exercised animals. Capillary density significantly increased in exercised animals, balancing cardiomyogenesis with neo-angiogenesis. c-kitpos eCSCs increased their number and activated state in exercising vs. sedentary animals. c-kitpos eCSCs in exercised hearts showed an increased expression of transcription factors, indicative of their commitment to either the cardiomyocyte (Nkx2.5pos) or capillary (Ets-1pos) lineages. These adaptations were dependent on exercise duration and intensity. Insulin-like growth factor-1, transforming growth factor-β1, neuregulin-1, bone morphogenetic protein-10, and periostin were significantly up-regulated in cardiomyocytes of exercised vs. sedentary animals. These factors differentially stimulated c-kitpos eCSC proliferation and commitment in vitro, pointing to a similar role in vivo. Conclusion Intensity-controlled exercise training initiates myocardial remodelling through increased cardiomyocyte growth factor expression leading to cardiomyocyte hypertrophy and to activation and ensuing differentiation of c-kitpos eCSCs. This leads to the generation of new myocardial cells. These findings highlight the endogenous regenerative capacity of the adult heart, represented by the eCSCs, and the fact that the physiological cardiac adaptation to exercise stress is a combination of cardiomyocyte hypertrophy and hyperplasia (cardiomyocytes and capillaries).

Catecholamine‐induced apoptosis and necrosis in cardiac and skeletal myocytes of the rat in vivo: the same or separate death pathways?

High levels of catecholamines are myotoxic but the relative amounts of apoptosis and necrosis have not been established in vivo in cardiac and skeletal muscles. Immunohistochemistry was used to detect and quantify myocyte‐specific necrosis (myosin antibody in vivo) and apoptosis (caspase‐3 antibody in vitro) in the heart and soleus muscles of male Wistar rats that had received single subcutaneous injections of isoprenaline over the range 1 μg to 5 mg [kg body weight (BW)]−1. Peak myocyte apoptosis occurred 3–6 h after, and necrosis 18 h after, a single injection of 5 mg (kg BW)−1 isoprenaline in vivo. In the heart myocyte death was mediated through the β1‐adrenergic receptor whereas myocyte death in the soleus muscle was mediated through the β2‐adrenergic receptor. Cardiomyocyte death was heterogeneously distributed throughout the heart, being greatest in the left ventricle (LV) subendocardium and peaking close to the apex, but with significantly more necrosis than apoptosis. Extensive co‐localization of caspase‐3 and myosin labelling was found in the myocytes of both the heart and the slow‐twitch soleus muscle. This, together with similar spatial distributions and responses to catecholamine doses, suggests that either caspase‐3 activation occurs in necrotic as well as apoptotic myocytes or that a large proportion of apoptotic myocytes progress to secondary necrosis in vivo.

Resident cardiac stem cells

Abstract.Regardless of erroneous claims by a minority of reports, adult cardiomyocytes are terminally differentiated cells which do not re-enter the cell-cycle under any known physiological or pathological circumstances. However, it has recently been shown that the adult heart has a robust myocardial regenerative potential, which challenges the accepted notions of cardiac cellular biology. The source of this regenerative potential is constituted by resident cardiac stem cells (CSCs). These CSCs, through both cell transplantation and in situ activation, have the capacity to regenerate significant segmental and diffuse myocyte losts, restoring anatomical integrity and ventricular function. Thus, CSC identification has started a brand new discipline of cardiac biology that could profoundly changed the outlook of cardiac physiology and the potential for treatment of cardiac failure. Nonetheless, the dawn of this new era should not be set back by premature attempts at clinical application before having accumulated the required scientifically reproducible data.

Concise Review: Heart Regeneration and the Role of Cardiac Stem Cells

Acute myocardial infarction leads to irreversible loss of cardiac myocytes, thereby diminishing the pump function of the heart. As a result, the strenuous workload imposed on the remaining cardiac myocytes often gives rise to subsequent cell loss until the vicious circle ends in chronic heart failure (CHF). Thus, we are in need of a therapy that could ameliorate or even reverse the disease progression of CHF. Endogenous regeneration of the mammalian heart has been shown in the neonatal heart, and the discovery that it may still persist in adulthood sparked hope for novel cardioregenerative therapies. As the basis for cardiomyocyte renewal, multipotent cardiac stem/progenitor cells (CSCs) that reside in the heart have been shown to differentiate into cardiac myocytes, smooth muscle cells, and vascular endothelial cells. These CSCs do have the potential to actively regenerate the heart but clearly fail to do so after abundant and segmental loss of cells, such as what occurs with myocardial infarction. Therefore, it is vital to continue research for the most optimal therapy based on the use or in situ stimulation of these CSCs. In this review, we discuss the current status of the cardioregenerative field. In particular, we summarize the current knowledge of CSCs as the regenerative substrate in the adult heart and their use in preclinical and clinical studies to repair the injured myocardium.