Adult c-kit(pos) 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.

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-kit(pos) eCSCs increased their number and activated state in exercising vs. sedentary animals. c-kit(pos) eCSCs in exercised hearts showed an increased expression of transcription factors, indicative of their commitment to either the cardiomyocyte (Nkx2.5(pos)) or capillary (Ets-1(pos)) lineages. These adaptations were dependent on exercise duration and intensity. Insulin-like growth factor-1, transforming growth factor-ß1, neuregulin-1, bone morphogenetic protein-10, and periostin were significantly up-regulated in cardiomyocytes of exercised vs. sedentary animals. These factors differentially stimulated c-kit(pos) 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-kit(pos) 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).

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.

Cardiac adaptations from 4 weeks of intensity-controlled vigorous exercise are lost after a similar period of detraining.

Intensity-controlled (relative to VO2max) treadmill exercise training in adult rats results in the activation and ensuing differentiation of endogenous c-kit(pos) cardiac stem/progenitor cells (eCSCs) into newly formed cardiomyocytes and capillaries. Whether these training-induced adaptations persist following detraining is undetermined. Twelve male Wistar rats (~230 g) were exercised at 80-85% of their VO2max for 30 min day(-1), 4 days week(-1) for 4 weeks (TR; n = 6), followed by 4 weeks of detraining (DTR; n = 6). Twelve untrained rats acted as controls (CTRL). Exercise training significantly enhanced VO2max (11.34 mL kg(-1) min(-1)) and wet heart weight (29%) above CTRL (P < 0.05). Echocardiography revealed that exercise training increased LV mass (~32%), posterior and septal wall thickness (~15%), ejection fraction and fractional shortening (~10%) compared to CTRL (P < 0.05). Cardiomyocyte diameter (17.9 ± 0.1 µm vs. 14.9 ± 0.6 µm), newly formed (BrdU(pos)/Ki67(pos)) cardiomyocytes (7.2 ± 1.3%/1.9 ± 0.7% vs. 0.2 ± 0.1%/0.1 ± 0.1%), total cardiomyocyte number (45.6 ± 0.6 × 10(6) vs. 42.5 ± 0.4 × 10(6)), c-kit(pos) eCSC number (884 ± 112 per 10(6) cardiomyocytes vs. 482 ± 132 per 10(6) cardiomyocytes), and capillary density (4123 ± 227 per mm(2) vs. 2117 ± 118 per mm(2)) were significantly greater in the LV of trained animals (P < 0.05) than CTRL. Detraining removed the stimulus for c-kit(pos) eCSC activation (640 ± 98 per 10(6) cardiomyocytes) and resultant cardiomyocyte hyperplasia (0.4 ± 0.3% BrdU(pos)/0.2 ± 0.2% Ki67(pos) cardiomyocytes). Capillary density (3673 ± 374 per mm(2)) and total myocyte number (44.7 ± 0.5 × 10(6)) remained elevated following detraining, but cardiomyocyte hypertrophy (15.0 ± 0.4 µm) was lost, resulting in a reduction of anatomical (wall thickness ~4%; LV mass ~10% and cardiac mass ~8%, above CTRL) and functional (EF & FS ~2% above CTRL) parameters gained through exercise training. These findings demonstrate that cardiac adaptations, produced by 4 weeks of intensity-controlled exercise training are lost after a similar period of detraining.

Isolation and characterization of resident endogenous c-Kit+ cardiac stem cells from the adult mouse and rat heart.

This protocol describes the isolation of endogenous c-Kit (also known as CD117)-positive (c-Kit(+)), CD45-negative (CD45(-)) cardiac stem cells (eCSCs) from whole adult mouse and rat hearts. The heart is enzymatically digested via retrograde perfusion of the coronary circulation, resulting in rapid and extensive breakdown of the whole heart. Next, the tissue is mechanically dissociated further and cell fractions are separated by centrifugation. The c-Kit(+)CD45(-) eCSC population is isolated by magnetic-activated cell sorting technology and purity and cell numbers are assessed by flow cytometry. This process takes ∼4 h for mouse eCSCs or 4.5 h for rat eCSCs. We also describe how to characterize c-Kit(+)CD45(-) eCSCs. The c-Kit(+)CD45(-) eCSCs exhibit the defining characteristics of stem cells: they are self-renewing, clonogenic and multipotent. This protocol also describes how to differentiate eCSCs into three main cardiac lineages: functional, beating cardiomyocytes, smooth muscle, and endothelial cells. These processes take 17-20 d.

Sustained delivery of insulin-like growth factor-1/hepatocyte growth factor stimulates endogenous cardiac repair in the chronic infarcted pig heart.

Activation of endogenous cardiac stem/progenitor cells (eCSCs) can improve cardiac repair after acute myocardial infarction. We studied whether the in situ activation of eCSCs by insulin-like growth factor 1 (IGF-1) and hepatocyte growth factor (HGF) could be increased using a newly developed hydrogel in chronic myocardial infarction (MI). One-month post-MI pigs underwent NOGA-guided intramyocardial injections of IGF-1/HGF (GF: both 0.5 µg/mL, n = 5) or IGF-1/HGF incorporated in UPy hydrogel (UPy-GF; both 0.5 µg/mL, n = 5). UPy hydrogel without added growth factors was administered to four control (CTRL) pigs. Left ventricular ejection fraction was increased in the UPy-GF and GF animals compared to CTRLs. UPy-GF delivery reduced pathological hypertrophy, led to the formation of new, small cardiomyocytes, and increased capillarization. The eCSC population was increased almost fourfold in the border zone of the UPy-GF-treated hearts compared to CTRL hearts. These results show that IGF-1/HGF therapy led to an improved cardiac function in chronic MI and that effect size could be further increased by using UPy hydrogel.

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 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.

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.

c-kitpos GATA-4 high rat cardiac stem cells foster adult cardiomyocyte survival through IGF-1 paracrine signalling.

BACKGROUND: Resident c-kit positive (c-kitpos) cardiac stem cells (CSCs) could be considered the most appropriate cell type for myocardial regeneration therapies. However, much is still unknown regarding their biological properties and potential. METHODOLOGY/PRINCIPAL FINDINGS: We produced clones of high and low expressing GATA-4 CSCs from long-term bulk-cultured c-kitpos CSCs isolated from adult rat hearts. When c-kitpos GATA-4 high expressing clonal CSCs (cCSCs) were co-cultured with adult rat ventricular cardiomyocytes, we observed increased survival and contractility of the cardiomyocytes, compared to cardiomyocytes cultured alone, co-cultured with fibroblasts or c-kitpos GATA-4 low expressing cCSCs. When analysed by ELISA, the concentration of IGF-1 was significantly increased in the c-kitpos GATA-4 high cCSC/cardiomyocyte co-cultures and there was a significant correlation between IGF-1 concentration and cardiomyocyte survival. We showed the activation of the IGF-1 receptor and its downstream molecular targets in cardiomyocytes co-cultured with c-kitpos GATA-4 high cCSCs but not in cardiomyocytes that were cultured alone, co-cultured with fibroblasts or c-kitpos GATA-4 low cCSCs. Addition of a blocking antibody specific to the IGF-1 receptor inhibited the survival of cardiomyocytes and prevented the activation of its signalling in cardiomyocytes in the c-kitpos GATA-4 high cCSC/cardiomyocyte co-culture system. IGF-1 supplementation or IGF-1 high conditioned medium taken from the co-culture of c-kitpos GATA-4 high cCSCs plus cardiomyocytes did extend the survival and contractility of cardiomyocytes cultured alone and cardiomyocytes co-cultured with c-kitpos GATA-4 low cCSCs. CONCLUSION/SIGNIFICANCE: c-kitpos GATA-4 high cCSCs exert a paracrine survival effect on cardiomyocytes through induction of the IGF-1R and signalling pathway.

Cardiac stem and progenitor cell identification: different markers for the same cell?

For a long time the heart has been considered a terminally differentiated organ without any regenerative potential. The latter has been classically based on the terminally differentiated nature of cardiomyocytes and the absence of a pool of tissue-specific stem cells. This view has been radically changed due to the discovery of resident cardiac stem and progenitor cells in the adult mammalian heart. However, at minimum, 5 apparently different cardiac stem and/or progenitor cell types have been described so far. Thus, we have changed from a view of the heart as a static tissue to an organ with the highest number of tissue-specific stem cell populations. Most likely, the different putative adult cardiac stem and progenitor cells represent different developmental and/or physiological stages of a unique resident adult cardiac stem cell. Notably, it is not yet known the origin of all these cells. A better understanding of the origin, biology and physiology of the myocardial stem and progenitor cells will impact the development of regenerative medicine as an effective therapy for heart disease and failure.