Cardiac Progenitor Research Articles

Thiamine increases resident endoglin positive cardiac progenitor cells and atrial contractile force in humans: A randomised controlled trial

BackgroundThe heart has an intrinsic ability to regenerate, orchestrated by progenitor or stem cells. However, the relative complexity of non-resident cardiac progenitor cell (CPC) therapy makes modulation of resident CPCs a more attractive treatment target. Thiamine analogues improve resident CPC function in pre-clinical models. In this double blinded randomised controlled trial (identifier: ACTRN12614000755639), we examined whether thiamine would improve CPC function in humans. Methods and resultsHigh dose oral thiamine (one gram twice daily) or matching placebo was administered 3–5 days prior to coronary artery bypass surgery (CABG). Right atrial appendages were collected at the time of CABG, and CPCs isolated. There was no difference in the primary outcome (proliferation ability of CPCs) between treatment groups. Older age was not associated with decreased proliferation ability. In exploratory analyses, isolated CPCs in the thiamine group showed an increase in the proportion of CD34−/CD105+ (endoglin) cells, but no difference in CD34−/CD90+ or CD34+ cells. Thiamine increased maximum force developed by isolated trabeculae, with no difference in relaxation time or beta-adrenergic responsiveness. ConclusionThiamine does not improve proliferation ability of CPC in patients undergoing CABG, but increases the proportion of CD34−/CD105+ cells. Having not met its primary endpoint, this study provides the impetus to re-examine CPC biology prior to any clinical outcome-based trial examining potential beneficial cardiovascular effects of thiamine.

The Spatiotemporal Expression of Notch1 and Numb and Their Functional Interaction during Cardiac Morphogenesis.

Numb family proteins (NFPs), including Numb and Numblike (Numbl), are commonly known for their role as cell fate determinants for multiple types of progenitor cells, mainly due to their function as Notch inhibitors. Previous studies have shown that myocardial NFP double knockout (MDKO) hearts display an up-regulated Notch activation and various defects in cardiac progenitor cell differentiation and cardiac morphogenesis. Whether enhanced Notch activation causes these defects in MDKO is not fully clear. To answer the question, we examined the spatiotemporal patterns of Notch1 expression, Notch activation, and Numb expression in the murine embryonic hearts using multiple approaches including RNAScope, and Numb and Notch reporter mouse lines. To further interrogate the interaction between NFPs and Notch signaling activation, we deleted both Notch1 or RBPJk alleles in the MDKO. We examined and compared the phenotypes of Notch1 knockout, NFPs double knockout, Notch1; Numb; Numbl and RBPJk; Numb; Numbl triple knockouts. Our study showed that Notch1 is expressed and activated in the myocardium at several stages, and Numb is enriched in the epicardium and did not show the asymmetric distribution in the myocardium. Cardiac-specific Notch1 deletion causes multiple structural defects and embryonic lethality. Notch1 or RBPJk deletion in MDKO did not rescue the structural defects in the MDKO but partially rescued the defects of cardiac progenitor cell differentiation, cardiomyocyte proliferation, and trabecular morphogenesis. Our study concludes that NFPs regulate progenitor cell differentiation, cardiomyocyte proliferation, and trabecular morphogenesis partially through Notch1 and play more roles than inhibiting Notch1 signaling during cardiac morphogenesis.

Upregulation of FoxM1 protects against ischemia/reperfusion-induced myocardial injury.

Ischemia/reperfusion (I/R) induced lethal tissue injury in myocardium. FoxM1 (Forkhead Box M1), expressed in proliferating cardiac progenitor cells, could regulate myocardial development. However, the role of FoxM1 in I/R-induced myocardial injury has not been reported yet. Rats were conducted with regional ischemia followed by reperfusion in myocardium through ligation of the left anterior descending coronary artery. Triphenyl-tetrazolium chloride staining was utilized to assess the infarct size. ELISA was performed to detect activities of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH). Protein expression of FoxM1 in heart tissues and H9c2 were determined by western blot. H9c2 cells were used to establish a hypoxia/reoxygenation cell model, and the cell viability, proliferation and apoptosis were evaluated by MTT, EdU (5-ethynyl-2'-deoxyuridine) staining and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining, respectively. Adenovirus (Ad)-mediated over-expression of FoxM1 was injected into the anterior wall of the left ventricle of rats to evaluate the role of FoxM1 on in vivo I/R-induced myocardial injury. FoxM1 was reduced in heart tissues isolated from rats post myocardial I/R injury. Forced FoxM1 expression increased cell viability and proliferation of hypoxia/reoxygenation-induced H9c2, while repressed the cell apoptosis with increased Bcl-2 and decreased Bax and cleaved caspase-3. Injection of Ad-FoxM1 suppressed infarct size of the heart and decreased activities of CK-MB and LDH. FoxM1 attenuated I/R-induced myocardial injury, providing potential therapeutic target for the disease.

Strategies for constructing pluripotent stem cell- and progenitor cell-derived three-dimensional cardiac micro-tissues.

Three-dimensional (3D) cardiac micro-tissue is a promising model for simulating the structural and functional features of heart in vitro. This scientific achievement provides a platform for exploration about the mechanisms on the development, damage, and regeneration of tissue, hence, paving a way toward development of novel therapies for heart diseases. However, 3D micro-tissue technology is still in its infant stages faced with many challenges such as incompleteness of the tissue microarchitecture, loss of the resident immune cells, poor reproducibility, and deficiencies in continuously feeding the nutrients and removing wastes during micro-tissue culturing. There is an urgent need to optimize the construction of 3D cardiac micro-tissue and improve functions of the involved cells. Therefore, scaffolds and cell resources for building 3D cardiac micro-tissues, strategies for inducing the maturation and functionalization of pluripotent stem cell- or cardiac progenitor cell-derived cardiomyocytes, and the major challenges were reviewed in this writing to enable future fabrication of 3D cardiac micro-tissues or organoids for drug screening, disease modeling, regeneration treatment, and so on.

Cell therapy using human cardiac progenitors cell-seeded collagen patches applied on failing overloaded right ventricle: A new step toward the treatment of right heart failure?

Right ventricular (RV) failure is a major concern in grown-up congenital heart diseases. Cell-based myocardial repair may be an innovative approach to restore overloaded RV function. A repaired Tetralogy of Fallot pig model was used. After 5 months, cell therapy was surgically administrated using human embryonic stem cell-derived Nkx2.5+ cardiac progenitor cell-seeded collagen patch. A large patch containing 10 7 cells was engineered to cover the whole RV free wall and applied on the epicardium of 4 pigs (treated group). A control group ( n = 4) received a cell-free patch (3) or no patch (1). Myocardial function was assessed by echocardiography using standard and strain parameters before and 2 months after cell graft. The fate of progenitors was tracked using human-and cardiac specific antibodies. Indexed RV diameter and telediastolic RV area significantly decreased in treated group whereas increased in control. The free wall thickness significantly increased in treated group compared to control. In the treated group, RV free wall strain significantly improved by contrast to control (respectively from −13.4 to −15.9% vs. −18.9 to −12.9%, P < 0.001). The global function was maintained in the treated group while it decreased in the control. Differentiating human cardiac progenitors were found in the patch and migrated within the myocardium ( Fig. 1 ). Although still immature, some cells featured TnT+ actinin+ sarcomeric units. Proliferating small Ki-67+ TnT+ labelled pig myocytes were observed. Expression of collagen genes col1a3, col3a1 monitored in real-Time QPCR reflecting myocardial fibrosis status significantly decreased in the treated group. Human cardiac progenitors migrated and differentiated, contributing together with triggered endogenous mechanism of repair to maintain the RV adaptation to overload and to improve free wall contractility. They also induced a proliferation of host cells supporting potential paracrine effects.

SDF-1 Molecularly Imprinted Biomimetic Scaffold as a Potential Strategy to Repair the Infarcted Myocardium

Background: In situ cardiac tissue engineering aims to heal the infarcted myocardium by guiding tissue regeneration within the patient body. A key step in this approach is the design of a bioactive scaffold, able to stimulate tissue repair at the site of damage. In the development of bioactive scaffolds, molecular imprinting nanotechnology has been recently proposed as a new functionalization strategy. Objectives: In this work, Molecularly Imprinted Particles (MIP) with recognition properties towards the stromal-derived factor-1 (SDF-1) were synthesized, characterized and used for the functionalization of a biomimetic scaffold. MIP are expected to favor the enrichment of the SDF-1 bioactive molecule within the scaffold, thereby promoting myocardial regeneration. Methods: MIP were obtained by precipitation polymerization, using the SDF-1 molecule as a template. Alginate/gelatin/elastin sponges were fabricated by freeze-drying and functionalized by MIP deposition. Morphological, physicochemical and functional analyses were performed both on MIP and on MIP-modified scaffolds. A preliminary biological in vitro investigation was also carried out using rat cardiac progenitor cells (rCPCs). Results: Imprinted nanoparticles with an average diameter between 0.6 and 0.9 µm were obtained. Infrared analysis of MIP confirmed the expected chemical structure. Recognition and selectivity tests showed that MIP were able to selectively recognize and rebind the template, even after their deposition on the scaffold. In vitro biological tests showed that cell adhesion to the scaffold was promoted by MIP functionalization. Conclusion: Results obtained in the present study suggest that biomimetic alginate/gelatin/elastin sponges, functionalized by MIP with recognition properties towards SDF-1, could be successfully used for tissue engineering approaches to repair the infarcted heart.

Functional siRNA Delivery by Extracellular Vesicle-Liposome Hybrid Nanoparticles.

The therapeutic use of RNA interference is limited by the inability of siRNA molecules to reach their site of action, the cytosol of target cells. Lipid nanoparticles, including liposomes, are commonly employed as siRNA carrier systems to overcome this hurdle, although their widespread use remains limited due to a lack of delivery efficiency. More recently, nature's own carriers of RNA, extracellular vesicles (EVs), are increasingly being considered as alternative siRNA delivery vehicles due to their intrinsic properties. However, they are difficult to load with exogenous cargo. Here, EV-liposome hybrid nanoparticles (hybrids) are prepared and evaluated as an alternative delivery system combining properties of both liposomes and EVs. It is shown that hybrids are spherical particles encapsulating siRNA, contain EV-surface makers, and functionally deliver siRNA to different cell types. The functional behavior of hybrids, in terms of cellular uptake, toxicity, and gene-silencing efficacy, is altered as compared to liposomes and varies among recipient cell types. Moreover, hybrids produced with cardiac progenitor cell (CPC) derived-EVs retain functional properties attributed to CPC-EVs such as activation of endothelial signaling and migration. To conclude, hybrids combine benefits of both synthetic and biological drug delivery systems and might serve as future therapeutic carriers of siRNA.

Strong as a Hippo’s Heart: Biomechanical Hippo Signaling During Zebrafish Cardiac Development

The heart is comprised of multiple tissues that contribute to its physiological functions. During development, the growth of myocardium and endocardium is coupled and morphogenetic processes within these separate tissue layers are integrated. Here, we discuss the roles of mechanosensitive Hippo signaling in growth and morphogenesis of the zebrafish heart. Hippo signaling is involved in defining numbers of cardiac progenitor cells derived from the secondary heart field, in restricting the growth of the epicardium, and in guiding trabeculation and outflow tract formation. Recent work also shows that myocardial chamber dimensions serve as a blueprint for Hippo signaling-dependent growth of the endocardium. Evidently, Hippo pathway components act at the crossroads of various signaling pathways involved in embryonic zebrafish heart development. Elucidating how biomechanical Hippo signaling guides heart morphogenesis has direct implications for our understanding of cardiac physiology and pathophysiology.

Pretreatment of cardiac progenitor cells with bradykinin attenuates H2O2-induced cell apoptosis and improves cardiac function in rats by regulating autophagy

BackgroundPrevious studies have demonstrated that human cardiac c-Kit+ progenitor cells (hCPCs) can effectively improve ischemic heart disease. However, the major challenge in applying hCPCs to clinical therapy is the low survival rate of graft hCPCs in the host heart, which limited the benefit of transplanted hCPCs. Bradykinin (BK) is a principal active agent of the tissue kinin-kallikrein system. Our previous studies have highlighted that BK mediated the growth and migration of CPCs by regulating Ca2+ influx. However, the protective effect of BK on CPCs, improvement in the survival rate of BK-pretreated hCPCs in the infarcted heart, and the related mechanism remain elusive.MethodsHCPCs were treated with H2O2 to induce cell apoptosis and autophagy, and different concentration of BK was applied to rescue the H2O2-induced injury detected by MTT assay, TUNEL staining, flow cytometry, western blotting, and mitoSOX assays. The role of autophagy in the anti-apoptotic effect of BK was chemically activated or inhibited using the autophagy inducer, rapamycin, or the inhibitor, 3-methyladenine (3-MA). To explore the protective effect of BK on hCPCs, 3-MA or BK-pretreated hCPCs were transplanted into the myocardial infarcted rats. An echocardiogram was used to determine cardiac function, H&E and Masson staining were employed to assess pathological characteristics, HLA gene expression was quantified by qRT-PCR, and immunostaining was applied to examine neovascularization using confocal microscopy.ResultsThe in vitro results showed that BK suppressed H2O2-induced hCPCs apoptosis and ROS production in a concentration-dependent manner by promoting pAkt and Bcl-2 expression and reducing cleaved caspase 3 and Bax expression. Moreover, BK restrained the H2O2-induced cell autophagy by decreasing LC3II/I, Beclin1, and ATG5 expression and increasing P62 expression. In the in vivo experiment, the transplanted BK- or 3-MA-treated hCPCs were found to be more effectively improved cardiac function by decreasing cardiomyocyte apoptosis, inflammatory infiltration, and myocardial fibrosis, and promoting neovascularization in the infarcted heart, compared to untreated-hCPCs or c-kit- cardiomyocytes (CPC- cells).ConclusionsOur present study established a new method to rescue transplanted hCPCs in the infarcted cardiac area via regulating cell apoptosis and autophagy of hCPCs by pretreatment with BK, providing a new therapeutic option for heart failure.

Transcriptional regulatory elements of hif1α in a distal locus of islet1 in Xenopus laevis.

Adult mammalian hearts are not regenerative. However, recent studies have evidenced that hypoxia enhances their regeneration. Islet1 (isl1) is known as a cardiac progenitor marker, which is quiescent in adult mammal hearts. In Xenopus hearts, transcriptional activation of isl1 was shown during cardiac regeneration of froglets at 3months after metamorphosis. In this study, we examined transcriptional regulation of isl1 focusing on hypoxia-inducible factor 1α (hif1α) in Xenopus heart. We found that hif1α expression was increased in response to cardiac injury and overexpression of hif1α upregulated mRNA expression of isl1. Multiple conservation analysis including 9 species revealed that 8 multiple conserved regions (MCRs) were present upstream of isl1. DNA sequence analysis using JASPAR showed hif1α binding motifs in MCRs. By luciferase reporter assay and chromatin immunoprecipitation analysis, we found that hif1α directly bound to hif1α motifs in the most distant MCR8 and showed a specific transcriptional activity on the MCR8. In the luciferase assay using constructs carrying MCR8 without a responsive motif of hif1α, the reporter activity was lost. Pharmacologically inhibition of hif1α affected isl1 transcription and downstream events including cardiac phenotypes, suggesting functional defects of islet1. Contrarily in murine hearts, transcription of isl1 was unresponsive even after cryoinjury to adult hearts while hif1α mRNA was induced. In comparative analysis of multiple alignment, hif1α elements present in MCR8 of Xenopus or zebrafish were found to be disrupted as species are evolutionarily distant from Xenopus and zebrafish. Our results suggested an altered switch of isl1 transcription between mammals and Xenopus laevis.

Expression dynamics of HAND1/2 in invitro human cardiomyocyte differentiation.

SummaryHand1 and Hand2 are transcriptional factors, and knockout mice of these genes show left and right ventricular hypoplasia, respectively. However, their function and expression in human cardiogenesis are not well studied. To delineate their expressions and assess their functions in human cardiomyocytes (CMs) in vitro, we established two triple-reporter human induced pluripotent stem cell lines that express HAND1mCherry, HAND2EGFP and either MYH6-driven iRFP670 or tagBFP constitutively and investigated their expression dynamics during cardiac differentiation. On day 5 of the differentiation, HAND1 expression marked cardiac progenitor cells. We profiled the CM subpopulations on day 20 with RNA sequencing and found that mCherry+ CMs showed higher proliferative ability than mCherry− CMs and identified a gene network of LEF1, HAND1, and HAND2 to regulate proliferation in CMs. Finally, we identified CD105 as a surface marker of highly proliferative CMs.

Unfathomed Nanomessages to the Heart: Translational Implications of Stem Cell-Derived, Progenitor Cell Exosomes in Cardiac Repair and Regeneration

Exosomes formed from the endosomal membranes at the lipid microdomains of multivesicular bodies (MVBs) have become crucial structures responsible for cell communication. This paracrine communication system between a myriad of cell types is essential for maintaining homeostasis and influencing various biological functions in immune, vasculogenic, and regenerative cell types in multiple organs in the body, including, but not limited to, cardiac cells and tissues. Characteristically, exosomes are identifiable by common proteins that participate in their biogenesis; however, many different proteins, mRNA, miRNAs, and lipids, have been identified that mediate intercellular communication and elicit multiple functions in other target cells. Although our understanding of exosomes is still limited, the last decade has seen a steep surge in translational studies involving the treatment of cardiovascular diseases with cell-free exosome fractions from cardiomyocytes (CMs), cardiosphere-derived cells (CDCs), endothelial cells (ECs), mesenchymal stromal cells (MSCs), or their combinations. However, most primary cells are difficult to culture in vitro and to generate sufficient exosomes to treat cardiac ischemia or promote cardiac regeneration effectively. Pluripotent stem cells (PSCs) offer the possibility of an unlimited supply of either committed or terminally differentiated cells and their exosomes for treating cardiovascular diseases (CVDs). This review discusses the promising prospects of treating CVDs using exosomes from cardiac progenitor cells (CPCs), endothelial progenitor cells (EPCs), MSCs, and cardiac fibroblasts derived from PSCs.

The role of bFGF in preventing the shrinkage of cardiac progenitor cell-engineered conduction tissue by downregulating α-SMA expression

AimsEngineered conduction tissues (ECTs) fabricated from cardiac progenitor cells (CPCs) and collagen sponges were precisely targeted for the treatment of atrioventricular conduction block in our previous studies. However, obvious shrinkage and deformation of ECTs was observed during in vitro culture. According to the literature, it can be speculated that basic fibroblast growth factor (bFGF) may downregulate alpha-smooth muscle actin (α-SMA) produced by CPCs to prevent the shrinkage of CPC-engineered conduction tissues. Main methodsIn this study, culture media with or without bFGF were used for both cell culture and 3D tissue construction. The expression of α-SMA and the size change of engineered tissue were analyzed to evaluate the feasibility of adding bFGF to regulate α-SMA expression and shrinkage of constructs. In addition, cardiac-specific examinations were performed to evaluate the effect of bFGF on cardiac tissue formation. Key findingsSupplementation with bFGF efficiently relieved shrinkage of engineered tissue by downregulating the expression of α-SMA at both the cellular and 3D tissue levels. Moreover, bFGF had a positive influence on cardiac tissue formation in terms of cell viability, tissue organization and electrical conduction velocity. SignificanceThis study provides a guide for both shape control and quality improvement of CPC-engineered cardiac tissues.

Hypoxia - as a Possible Regulator of the Activity of Epicardial Mesothelial Cells After Myocardial Infarction.

Aim To study the effect of hypoxia on the activity of epithelial-mesenchymal transition (EMT) in epicardial cells, which provides formation of a specialized microenvironment.Material and methods This study used a model of experimental myocardial infarction created by ligation of the anterior descendent coronary artery. The activity of epicardial cells after a hypoxic exposure was studied with the hypoxia marker, pimonidazole, bromodeoxyuridine, immunofluorescent staining of heart cryosections, and in vitro mesothelial cell culture.Results The undamaged heart maintained the quiescent condition of mesothelial cells and low levels of their proliferation, extracellular matrix protein production, and of the EMT activity. Acute ischemic injury induced moderate hypoxia in the epicardial/subepicardial region. This caused a global rearrangement of this region due to the initiation of EMT in cells, changes in the cell composition, and accumulation of extracellular matrix proteins. We found that the initiation of EMT in mesothelial cells may result in the formation of smooth muscle cell precursors, fibroblasts, and a population of Sca-1+ cardiac progenitor cells, which may both participate in construction of new blood vessels and serve as a mesenchymal link for the paracrine support of microenvironmental cells. In in vitro experiments, we showed that 72‑h hypoxia facilitated activation of EMT regulatory genes, induced dissembling of intercellular contacts, cell uncoupling, and increased cell plasticity.Conclusion The epicardium of an adult heart serves as a "reparative reserve" that can be reactivated by a hypoxic exposure. This creates a basis for an approach to influence the epicardium to modulate its activity for regulating reparative processes.

Applications of Omics Technologies in Regenerative Therapies for Cardiac Repair

Lately, cardio regenerative therapeutic approaches in clinic have received strong boost from promising research advances in stem cells. Researchers have shown applications of the first generation of clinical trials using cell-based therapies in the heart that have been performed with bone marrow and adipose tissue derived mesenchymal stem cells shortcomings in the first generation cells. Second generation cell therapies are considered superior and being considered towards the use of cardiac-committed cell populations. These include cardiac progenitor cells and pluripotent stem cell derived cardiomyocytes. However, translating the research laboratory results along with pre-clinical data into effective clinical treatments is still thought to be challenging. This is due to the existing lack of knowledge on the regenerative mechanisms of action of these therapeutic products in addition to the stringent regulatory and safety concerns. This has prompted researchers to consider advanced analytical methods for characterization of such complex products and a deep understanding of their therapeutic effects at the cell and molecular level. This characterization is considered very critical to overcome the challenges and make these cells compatible with the advanced therapies. To this end, omics technologies including proteomics and glyco(proteo)mics that are based on state-of-the-art mass-spectrometry have shown tremendous promise to generate novel data on cell biology along with the required assessment of cell based-products that are applied in cardiac regeneration strategies. In this perspective, we have described proteomics and glyco(proteo)mics and discuss the impact of omics technologies on cardiac progenitor cells and pluripotent stem cell derived cardiomyocytes biology cardiac regenerative therapies.

Training biochemistry students in experimental developmental biology: Induction of cardia bifida formation in the chick embryo.

A high variety of experimental model organisms have been used in developmental biology practical lectures. The work with developing embryos is crucial to make students aware of the multiple biological phenomena underlying normal animal embryogenesis and morphogenesis and represent a unique experimental platform to analyze the impact of molecular signaling in the regulation of all these processes. In particular, Biochemistry undergraduate students enjoy both practical and theoretical lectures on the molecular mechanisms of embryonic development, as that allows them for the integration of crucial molecular concepts (e.g. signaling and signal transduction mechanisms; molecular patterning of development) into the dynamic and progressive context of animal embryonic ontogenesis. Accordingly, it is important to carefully design practical laboratory lectures in developmental biology, as these are a unique pedagogical tools fostering the interests of the students in this subject. This study describes the design, implementation, and evaluation of a two-session laboratory practical activity performed by Biochemistry undergraduate students at University of Málaga (Spain). In this practical activity, which takes advantage of the unique characteristics of the chick embryo, students learn how the vertebrate heart forms from the fusion of two bilateral-symmetric cardiac progenitor pools under the guidance of the underlying endoderm. This cheap and easy practical laboratory activity provides relevant visual information on how experimental manipulations can severely influence anatomical form during organ development, as well as an excellent experimental setting to test molecular regulation of morphogenesis in an ex vivo (ex ovo) context.

Direct Reprogramming of Cardiac Fibroblasts to Repair the Injured Heart.

Coronary heart disease is a leading cause of mortality and morbidity. Those that survive acute myocardial infarction are at significant risk of subsequent heart failure due to fibrotic remodelling of the infarcted myocardium. By applying knowledge from the study of embryonic cardiovascular development, modern medicine offers hope for treatment of this condition through regeneration of the myocardium by direct reprogramming of fibrotic scar tissue. Here, we will review mechanisms of cell fate specification leading to the generation of cardiovascular cell types in the embryo and use this as a framework in which to understand direct reprogramming. Driving expression of a network of transcription factors, micro RNA or small molecule epigenetic modifiers can reverse epigenetic silencing, reverting differentiated cells to a state of induced pluripotency. The pluripotent state can be bypassed by direct reprogramming in which one differentiated cell type can be transdifferentiated into another. Transdifferentiating cardiac fibroblasts to cardiomyocytes requires a network of transcription factors similar to that observed in embryonic multipotent cardiac progenitors. There is some flexibility in the composition of this network. These studies raise the possibility that the failing heart could one day be regenerated by directly reprogramming cardiac fibroblasts within post-infarct scar tissue.

HCN4 current during human sinoatrial node-like action potentials

BackgroundDespite the many studies carried out over the past 40 years, the contribution of the HCN4 encoded hyperpolarization-activated ‘funny’ current (If) to pacemaker activity in the mammalian sinoatrial node (SAN), and the human SAN in particular, is still controversial and not fully established. ObjectiveTo study the contribution of If to diastolic depolarization of human SAN cells and its dependence on heart rate, cAMP levels, and atrial load. MethodsHCN4 channels were expressed in human cardiac myocyte progenitor cells (CMPCs) and HCN4 currents assessed using perforated patch-clamp in traditional voltage clamp mode and during action potential clamp with human SAN-like action potential waveforms with 500–1500 ms cycle length, in absence or presence of forskolin to mimic β-adrenergic stimulation and a −15 mV command potential offset to mimic atrial load. ResultsForskolin significantly increased the fully-activated HCN4 current density at −140 mV by 14% and shifted the steady-state activation curve by +7.4 mV without affecting its slope. In addition, forskolin significantly accelerated current activation but slowed deactivation. The HCN4 current did not completely deactivate before the subsequent diastolic depolarization during action potential clamp. The amplitude of HCN4 current increased with increasing cycle length, was significantly larger in the presence of forskolin at all cycle lengths, and was significantly increased upon the negative offset to the command potential. ConclusionsIf is active during a human SAN action potential waveform and its amplitude is modulated by heart rate, β-adrenergic stimulation, and diastolic voltage range, such that If is under delicate control.

Doxorubicin induces an alarmin-like TLR4-dependent autocrine/paracrine action of Nucleophosmin in human cardiac mesenchymal progenitor cells

BackgroundDoxorubicin (Dox) is an anti-cancer anthracycline drug that causes double-stranded DNA breaks. It is highly effective against several types of tumours; however, it also has adverse effects on regenerative populations of normal cells, such as human cardiac mesenchymal progenitor cells (hCmPCs), and its clinical use is limited by cardiotoxicity. Another known effect of Dox is nucleolar disruption, which triggers the ubiquitously expressed nucleolar phosphoprotein Nucleophosmin (NPM) to be released from the nucleolus into the cell, where it participates in the orchestration of cellular stress responses. NPM has also been observed in the extracellular space in response to different stress stimuli; however, the mechanism behind this and its functional implications are as yet largely unexplored. The aim of this study was to establish whether Dox could elicit NPM secretion in the extracellular space and to elucidate the mechanism of secretion and the effect of extracellular NPM on hCmPCs.ResultsWe found that following the double-strand break formation in hCmPCs caused by Dox, NPM was rapidly secreted in the extracellular space by an active mechanism, in the absence of either apoptosis or necrosis. Extracellular release of NPM was similarly seen in response to ultraviolet radiation (UV). Furthermore, we observed an increase of NPM levels in the plasma of Dox-treated mice; thus, NPM release also occurred in vivo. The treatment of hCmPCs with extracellular recombinant NPM induced a decrease of cell proliferation and a response mediated through the Toll-like receptor (TLR)4. We demonstrated that NPM binds to TLR4, and via TLR4, and nuclear factor kappa B (NFkB) activation/nuclear translocation, exerts proinflammatory functions by inducing IL-6 and COX-2 gene expression. Finally, we found that in hCmPCs, NPM secretion could be driven by an autophagy-dependent unconventional mechanism that requires TLR4, since TLR4 inhibition dramatically reduced Dox-induced secretion.ConclusionsWe hypothesise that the extracellular release of NPM could be a general response to DNA damage since it can be elicited by either a chemical agent such as Dox or a physical genotoxic stressor such as UV radiation. Following genotoxic stress, NPM acts similarly to an alarmin in hCmPCs, being rapidly secreted and promoting cell cycle arrest and a TLR4/NFκB-dependent inflammatory response.

374-P: Oleate Prevents Palmitate-Induced Abnormalities in Insulin Signaling in Human Cardiac Progenitor Cells

Myocardial ectopic lipid deposition has been associated with insulin resistance and cardiovascular risk. Palmitate, one of the most abundant saturated fatty acids, has been shown to impair insulin signaling in multiple cell types. Human cardiac progenitor cells (CPC) represent a compartment of multipotent stem cells essential for constant tissue repair and renewal in the adult heart. In this study, the ability of palmitate to impair insulin signaling, and the modulatory effects of oleate, a mono-unsaturated fatty acid, on palmitate-induced abnormalities were investigated in human CPC isolated from control subjects undergoing cardiac surgery. Human CPC express insulin receptor (IR), as assessed by quantitative RT-PCR and immunoblotting. Specifically, in human CPC both IR isoforms, A (IR-A) and B (IR-B), were identified, and IR-A was more expressed than IR-B. Exposure of human CPC to 100 nM insulin for 15 minutes resulted in increased Akt and p44/p42 MAPK phosphorylation (p&amp;lt;0.05), evidenced by immunoblotting. Treatment of human CPC with 0.25 mM palmitate for 24 h impaired the ability of insulin, added for the last 15 minutes, to phosphorylate both Akt and p44/p42 MAPK (p&amp;lt;0.05), without no effect on total Akt and p44/p42 MAPK protein levels. Interestingly, palmitate was able to increase both IR-A and IR-B mRNA levels (p&amp;lt;0.05), and to reduce IR protein expression (p&amp;lt;0.05). Co-incubation of palmitate with 0.1 mM oleate prevented the ability of palmitate to reduce insulin-induced Akt phosphorylation (p&amp;lt;0.05), but did not rescue the impaired p44/p42 MAPK phosphorylation. In addition, co-treatment with oleate also prevented both palmitate-induced upregulation of IR-A and IR-B mRNA (p&amp;lt;0.05), and downregulation of IR protein levels (p&amp;lt;0.05). In conclusion, oleate prevents some of the palmitate-induced impairments in insulin signaling in human CPC, thus contributing to cardiac protection from lipotoxicity-induced metabolic alterations. Disclosure R. Doria: None. F. Giorgino: Consultant; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Lilly Diabetes, Novo Nordisk, Roche Diabetes Care, Sanofi, Research Support; Self; Lilly Diabetes, Roche Diabetes Care. C. Caccioppoli: None. R. Schipani: None. V. Genchi: None. A. Leonardini: None. A. Natalicchio: Speaker’s Bureau; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Lilly Diabetes, Sanofi. S. Perrini: None. A. Cignarelli: None. L. Laviola: Advisory Panel; Self; A. Menarini Diagnostics, Abbott, AstraZeneca, Lilly Diabetes, Novo Nordisk Inc., Roche Diabetes Care, Sanofi-Aventis, Speaker’s Bureau; Self; Boehringer Ingelheim International GmbH, Medtronic.