Endogenous Cardiac Progenitor Cells Research Articles

Stem cell exosomes in the pathophysiology of cardiovascular diseases

This study focused on the current state of the therapeutic potential of extracellular vesicles, which depends on the methods of their isolation and composition and the characteristics of the vesicular and nonvesicular components. Myocardial damage, particularly as a result of acute myocardial infarction, leads to irreversible death of cardiomyocytes and sarcomeres and ultimately to heart failure. The adult heart has limited regenerative capacity; therefore, stimulation of endogenous repair and regenerative potentials using cell therapy has potential. Moreover, the benefit from the injection of stem cells and progenitor cells into the damaged myocardium is mediated by the factors they secrete. In particular, exosomes, nanosized secreted extracellular vesicles of endosomal origin, have become key signaling organelles in intercellular communication and are currently considered key regenerative components of the secretome of stem and progenitor cells. Exosomes released from cardiac embryonic and mesenchymal stem cells, resident stem and progenitor cells (including a specific subgroup of cardiosphere cells), induced pluripotent stem cells, and cardiomyocytes isolated from these cells have cardioprotective, immunomodulatory, and reparative abilities. The use of exosomes in the targeted transport of drugs in lipid-like nanocontainers and extracellular vesicles is another promising area. Because artificial drug carriers, including liposomes and lipid-based nanoparticles, are limited by potential toxicity, immunogenicity, and inability to target specific organs, exosomes hold good promise as potential drug carriers. Compounds can be transported both inside exosomes and on their surface. Secreted extracellular vesicles, particularly exosomes, can be considered a key functional component of the secretome of stem cells and cardiogenic progenitor cells (mesenchymal stem cells, endogenous cardiac progenitor cells, cardiospheres, bone marrow embryonic stem cells, and bone marrow induced pluripotent stem cells). They have demonstrated therapeutic efficacy in preclinical models in the study of cardiovascular pathology.

Identification of SALL4 Expressing Islet-1+ Cardiovascular Progenitor Cell Clones.

The utilization of cardiac progenitor cells (CPCs) has been shown to induce favorable regenerative effects. While there are various populations of endogenous CPCs in the heart, there is no consensus regarding which population is ideal for cell-based regenerative therapy. Early-stage progenitor cells can be differentiated into all cardiovascular lineages, including cardiomyocytes and endothelial cells. Identifying an Islet-1+ (Isl-1+) early-stage progenitor population with enhanced stemness, multipotency and differentiation potential would be beneficial for the development of novel regenerative therapies. Here, we investigated the transcriptome of human neonatal Isl-1+ CPCs. Isl-1+ human neonatal CPCs exhibit enhanced stemness properties and were found to express Spalt-like transcription factor 4 (SALL4). SALL4 plays a role in embryonic development as well as proliferation and expansion of hematopoietic progenitor cells. SALL4, SOX2, EpCAM and TBX5 are co-expressed in the majority of Isl-1+ clones isolated from neonatal patients. The pre-mesendodermal transcript TFAP2C was identified in select Isl-1, SALL4, SOX2, EpCAM and TBX5 expressing clones. The ability to isolate and expand pre-mesendodermal stage cells from human patients is a novel finding that holds potential value for applications in regenerative medicine.

Discovery of retinoic acid receptor agonists as proliferators of cardiac progenitor cells through a phenotypic screening approach.

Identification of small molecules with the potential to selectively proliferate cardiac progenitor cells (CPCs) will aid our understanding of the signaling pathways and mechanisms involved and could ultimately provide tools for regenerative therapies for the treatment of post‐MI cardiac dysfunction. We have used an in vitro human induced pluripotent stem cell‐derived CPC model to screen a 10,000‐compound library containing molecules representing different target classes and compounds reported to modulate the phenotype of stem or primary cells. The primary readout of this phenotypic screen was proliferation as measured by nuclear count. We identified retinoic acid receptor (RAR) agonists as potent proliferators of CPCs. The CPCs retained their progenitor phenotype following proliferation and the identified RAR agonists did not proliferate human cardiac fibroblasts, the major cell type in the heart. In addition, the RAR agonists were able to proliferate an independent source of CPCs, HuES6. The RAR agonists had a time‐of‐differentiation‐dependent effect on the HuES6‐derived CPCs. At 4 days of differentiation, treatment with retinoic acid induced differentiation of the CPCs to atrial cells. However, after 5 days of differentiation treatment with RAR agonists led to an inhibition of terminal differentiation to cardiomyocytes and enhanced the proliferation of the cells. RAR agonists, at least transiently, enhance the proliferation of human CPCs, at the expense of terminal cardiac differentiation. How this mechanism translates in vivo to activate endogenous CPCs and whether enhancing proliferation of these rare progenitor cells is sufficient to enhance cardiac repair remains to be investigated.

Triggered Cardiogenesis in Wingless 5a Treated Amniotic Mesenchymal Stromal Cells

Adult cardiac myocytes are understood to be terminally differentiated. Although several reports have highlighted the presence of endogenous cardiac progenitor cells, an ongoing debate exits on their origin and role in myocardial regeneration [1,2].

Changes in coexpression of pericytes and endogenous cardiac progenitor cells from heart development to disease state.

Focussing on the potential role of cardiovascular cell therapy, we investigated the spatial relationship between pericytes (cells with cardiac repair capabilities that ensheath blood vessels) and endogenous cardiac progenitors within stem cells' niches. We explored possible changes in their co-localisation in developing human hearts from foetal to adult stage and following ischaemia. Foetal and adult human heart specimens, obtained under ethical consent (University of Edinburgh ethics committee), were used for immunohistochemistry, cell isolation, culture and differentiation. Multi-lineage differentiation in culture, by single and double staining was completed for CD 146+ foetal pericytes and c-kit+ cells. Endothelial markers (CD31) gene expression was quantified by qPCR. c-kit+ cells frequency and coexpression with pericytes decrease with heart development, already evident by gestation week 19th. Pericytes and c-kit+ cells express the early cardiac transcription factors Nkx2.5 and Islet 1. Only c-kit+ cells express the stemness marker SSEA3 (24%), known to progressively decrease with cell differentiation. Endothelial differentiation assessment shows that cardiac pericytes and c-kit+ cells do not form CD31+ networks. This finding correlates with absence of staining for CD31 marker in both cultured cells' types. The cardiac marker α-actin was present in both cell populations. In healthy adult heart, pericyte markers CD146 localise within the vasculature. Following ischaemia this pericyte marker becomes also evident outside the vasculature.In healthy adult atrium, c-kit expression is low and coexpression with other markers inconspicuous. Ischaemia leads to increased c-kit expression, particularly in blood vessels <50um diameter. Furthermore, following ischaemia c-kit, endothelium and pericyte markers co-localise within the same atrial cells. Blood vessels >50μm diameter showed mostly only staining for endothelial (vWF) and pericyte (CD146) markers, with no co-expression of c-kit marker identified. Staining patterns within the ischaemic regions of the right and left atrial appendages revealed low levels of colocalisation between vWF and CD146. Acute ischaemia of the left ventricle affected the detection of cardiac stem cells markers in the area of injury, due to myocardium disruption. Foetal heart pericytes and c-kit+ cells express early cardiac transcription factors and show trans-differentiation potential, which decreases in healthy adult hearts. The preservation and activity of cardiac stem cells niches within the atrium vasculature appears re-activated in post-ischaemic hearts. Better understanding of cardiac c-kit+ and pericyte cells during-human embryonic development and during ischaemia may identify alternative novel therapeutic strategy against coronary artery disease.

Calcium-dependent potassium channels control proliferation of cardiac progenitor cells and bone marrow-derived mesenchymal stem cells.

Ex vivo proliferated c-Kit+ endogenous cardiac progenitor cells (eCPCs) obtained from mouse and human cardiac tissues have been reported to express a wide range of functional ion channels. In contrast to previous reports in cultured c-Kit+ eCPCs, we found that ion currents were minimal in freshly isolated cells. However, inclusion of free Ca2+ intracellularly revealed a prominent inwardly rectifying current identified as the intermediate conductance Ca2+ -activated K+ current (KCa3.1) Electrical function of both c-Kit+ eCPCs and bone marrow-derived mesenchymal stem cells is critically governed by KCa3.1 calcium-dependent potassium channels. Ca2+ -induced increases in KCa3.1 conductance are necessary to optimize membrane potential during Ca2+ entry. Membrane hyperpolarization due to KCa3.1 activation maintains the driving force for Ca2+ entry that activates stem cell proliferation. Cardiac disease downregulates KCa3.1 channels in resident cardiac progenitor cells. Alterations in KCa3.1 may have pathophysiological and therapeutic significance in regenerative medicine. Endogenous c-Kit+ cardiac progenitor cells (eCPCs) and bone marrow (BM)-derived mesenchymal stem cells (MSCs) are being developed for cardiac regenerative therapy, but a better understanding of their physiology is needed. Here, we addressed the unknown functional role of ion channels in freshly isolated eCPCs and expanded BM-MSCs using patch-clamp, microfluorometry and confocal microscopy. Isolated c-Kit+ eCPCs were purified from dog hearts by immunomagnetic selection. Ion currents were barely detectable in freshly isolated c-Kit+ eCPCs with buffering of intracellular calcium (Ca2+i ). Under conditions allowing free intracellular Ca2+ , freshly isolated c-Kit+ eCPCs and ex vivo proliferated BM-MSCs showed prominent voltage-independent conductances that were sensitive to intermediate-conductance K+ -channel (KCa3.1 current, IKCa3.1 ) blockers and corresponding gene (KCNN4)-expression knockdown. Depletion of Ca2+i induced membrane-potential (Vmem ) depolarization, while store-operated Ca2+ entry (SOCE) hyperpolarized Vmem in both cell types. The hyperpolarizing SOCE effect was substantially reduced by IKCa3.1 or SOCE blockade (TRAM-34, 2-APB), and IKCa3.1 blockade (TRAM-34) or KCNN4-knockdown decreased the Ca2+ entry resulting from SOCE. IKCa3.1 suppression reduced c-Kit+ eCPC and BM-MSC proliferation, while significantly altering the profile of cyclin expression. IKCa3.1 was reduced in c-Kit+ eCPCs isolated from dogs with congestive heart failure (CHF), along with corresponding KCNN4 mRNA. Under perforated-patch conditions to maintain physiological [Ca2+ ]i , c-Kit+ eCPCs from CHF dogs had less negative resting membrane potentials (-58±7mV) versus c-Kit+ eCPCs from control dogs (-73±3mV, P<0.05), along with slower proliferation. Our study suggests that Ca2+ -induced increases in IKCa3.1 are necessary to optimize membrane potential during the Ca2+ entry that activates progenitor cell proliferation, and that alterations in KCa3.1 may have pathophysiological and therapeutic significance in regenerative medicine.

Qualitative and Quantitative Analysis of Cardiac Progenitor Cells in Cases of Myocarditis and Cardiomyopathy.

We aimed to identify and quantify CD117+ and CD90+ endogenous cardiac progenitor cells (CPC) in human healthy and diseased hearts. We hypothesize that these cells perform a locally acting, contributing function in overcoming medical conditions of the heart by endogenous means. Human myocardium biopsies were obtained from 23 patients with the following diagnoses: Dilatative cardiomyopathy (DCM), ischemic cardiomyopathy (ICM), myocarditis, and controls from healthy cardiac patients. High-resolution scanning microscopy of the whole slide enabled a computer-based immunohistochemical quantification of CD117 and CD90. Those signals were evaluated by Definiens Tissue Phenomics® Technology. Co-localization of CD117 and CD90 was determined by analyzing comparable serial sections. CD117+/CD90+ cardiac cells were detected in all biopsies. The highest expression of CD90 was revealed in the myocarditis group. CD117 was significantly higher in all patient groups, compared to healthy specimens (*p < 0.05). The highest co-expression was found in the myocarditis group (6.75 ± 3.25 CD90+CD117+ cells/mm2) followed by ICM (4 ± 1.89 cells/mm2), DCM (1.67 ± 0.58 cells/mm2), and healthy specimens (1 ± 0.43 cells/mm2). We conclude that the human heart comprises a fraction of local CD117+ and CD90+ cells. We hypothesize that these cells are part of local endogenous progenitor cells due to the co-expression of CD90 and CD117. With novel digital image analysis technologies, a quantification of the CD117 and CD90 signals is available. Our experiments reveal an increase of CD117 and CD90 in patients with myocarditis.

Abstract 21: Ploidy Alteration of Murine Cardiac Progenitor Cells in Response to Infarction Injury

Introduction: Discovery of endogenous cardiac progenitor cells (CPC) prompted intense research efforts in multiple experimental animal models and clinical trials for heart failure treatment. Our lab identified a fundamental difference in ploidy content between rodent (rat, mouse) CPCs possessing mononuclear tetraploid (4n) chromosome content versus large mammal (human, swine) CPCs with mononuclear diploid (2n) content. Ploidy differences raise provocative questions regarding translational applicability of myocardial regeneration in rodents as polyplodization often correlates with enhanced regenerative potential. Hypothesis: Mononuclear chromatin duplication in CPCs improves regenerative capacity of the heart through higher stress resistance and overriding senescence cell-cycle arrest. Methods and Results: Ploidy of cultured CPCs is consistent and stable ploidy content over increased passages with samples from eight humans, two swine strains, six mouse strains, and seven rat clonal lines as determined by karyotype, confocal microscope and flow cytometry analyses. In situ ploidy analysis of CPCs reveals diploid content in human tissue and a mixture of mononuclear diploid and tetraploid nuclei in mouse, confirmed using freshly isolated Lin- c-kit+ CPCs. Tetraploid nuclear phenotype of murine CPCs is markedly different from predominantly diploid (2n) murine c-kit+ cells located in other tissues such as intestine and bone marrow. Higher ploidy content concurrent with expansion of the CPC pool are evident in the border zone at seven days post-infarction in adult FVB mice compared to age and gender matched non-injured hearts. Conclusion: Tetraploid c-kit+ cells found within the rodent heart may contribute to species-specific characteristics of stem cells and myocardial regenerative capacity. Future studies will focus upon the biological properties of diploid versus tetraploid CPCs and advantages of polyploid content for mediating myocardial regeneration.

PP.31.10] ENDOTHELIAL CELL GROWTH SUPPLEMENT IMPROVES CARDIAC FUNCTION FOLLOWING MOUSE MYOCARDIAL INFARCTION

Objective: Myocardial infarction (MI) and subsequent heart failure is a leading cause of death in high or middle income countries. Development of treatments that stimulate heart tissue regeneration and revascularization/angiogenesis is important to improve the prognosis of these patients. Our recent work demonstrated that endothelial cell growth supplement (ECGS)-containing medium stimulates endothelial differentiation of c-kit positive cardiac progenitor cells in vitro. The objective of the current work was to test whether ECGS can activate endogenous cardiac progenitor cells and improve cardiac function in a mouse MI model. Design and method: Ligation of the left anterior descending (LAD) artery was performed on mice at 2 months of age under anesthetization and intubation. After displacing the pericardium, a 7–0 silk suture was passed under the LAD artery 2 mm below the left atrium and permanently tied, to eliminate blood flow to the area distal to the suture. ECGS or saline (as control) were injected intramyocardially to the ligation area through a sterile Hamilton syringe with a 30 gauge needle immediately after the ligation. Mice that survived the procedure were maintained for 3 weeks. Echocardiography was performed to monitor the cardiac function prior to organ collection. Results: The results showed that MI increased c-kit positive cells and blood vessel formation as well as massive fibrosis in the infarcted area 3 weeks post-surgery. ECGS treatment significantly reduced cardiac fibrosis, especially in the non-infarcted area and border zone (1.96 ± 0.28 % in ECGS treated group vs 4.54 ± 1.08 % in saline treated control group, p < 0.05). ECGS also improved ejection fraction in these animals compared to the saline treated control group (49.9 ± 2.9 % ECGS treated vs 37.4 ± 4.6 % saline treated, p < 0.05). However, ECGS had no significant effect on infarct size in these animals. Conclusions: The current study demonstrates a reduction in cardiac fibrosis and improvement of cardiac function with ECGS in a mouse MI model, which may link to our in vitro observation that ECGS can activate endothelial differentiation of cardiac progenitor cells.

Abstract 23: p53 Mediates de novo Cardiomyocte Differentiation From c-kit Positive Cardiac Progenitor Cells

The heart is arguably the least regenerative organ. Nonetheless, cardiac progenitor cells (CPCs) can readily be isolated from the adult heart. Moreover, we recently demonstrated that c-kit+ CPCs are able to generate de novo lineages of cardiomyocytes, endothelial cells and fibroblasts, albeit at low levels. In the present study, we assess if different pathophysiological stimuli could enhance cardiomyocyte lineage differentiation by CPCs. We first established that CPCs are heterogeneous in vivo and can be classified into three main populations based on the respective gene expression profiles: uncommitted, vascular and cardiogenic CPCs. Next, we show that transverse aortic constriction (TAC) surgery increased CPC derived cardiomyocytes by 3-fold, and enhanced non-cardiomyocyte lineages as well. Interestingly, anthracycline-induced cardiomyopathy increased CPC-derived cardiomyocytes by 35-fold. Immunostaining showed that doxorubicin stimulates p53 expression within CPCs and selectively enhanced CPC-derived cardiomyocyte lineage. The selective p53 inhibitor, pifithrin α, completely blocked the doxorubicin-mediated increase in de novo cardiomyocyte formation. Administration of p53 Activator III, RITA (reactivation of p53 and induction of tumor cell apoptosis), was sufficient to induce CPC-derived cardiomyocyte differentiation. These results demonstrate that anthracyclines can increase de novo cardiomyocyte differentiation from CPCs through activation of the p53 pathway. Ultimately, these findings might lead to new therapies for cardiac regeneration by inducing cardiomyocyte differentiation by endogenous CPCs.

Abstract 218: Cardiac Side Population Cells Give Rise to Cardiomyocytes in the Adult Heart

Therapies that enhance cardiac regeneration have the potential to improve heart failure outcomes. One strategy to enhance cardiac regeneration is by activating endogenous cardiac progenitor cells. Cardiac side population cells (cSPCs) are proposed progenitor cells that reside in the heart; however, their putative progenitor cell properties are based on cell culture data and transplantation studies performed with isolated cSPCs. To determine the endogenous regenerative potential of cSPCs in vivo , we generated a mouse model that harbors an Abcg2-driven, tamoxifen-inducible Cre recombinase and crossed it to a GFP reporter mice. One month after tamoxifen treatment, we obtained efficient GFP-labeling of side population cells with 47.0 ± 11.05% of bone marrow side population cells and 75.8 ± 10.87% of cSPCs. Importantly, during a one-month chase period, we observed a three-fold increase in GFP-labeled cardiomyocytes when compared to a 1-week chase period. We quantified the extent of GFP-labeling of cardiomyocytes using adult cardiomyocyte isolation, where we measured 0.8% GFP labeled cardiomyocytes after a one-month chase period. We also observed labeling of many other cell types in the heart, such as endothelial cells, smooth muscle cells, and pericytes. We are currently testing to what extent cardiac injury enhances cSPC derived cardiomyocyte formation. Using our mouse model that efficiently labels side population cells in vivo , we demonstrated that cSPCs contribute cardiomyocytes in the adult heart in vivo .

Abstract 126: Triptonide is an Effective Inhibitor of Wnt Signaling

Canonical Wnt/β-catenin signaling plays important roles during vertebrate heart development and inhibition of this pathway has been shown to improve injured heart repair by promoting cardiac progenitor cells’ differentiation. Despite extensive research, to date, no approved Wnt inhibitors are available in clinic. Triptonide is a key bioactive small molecule identified in a traditional Chinese medicine named Tripterygium wilfordii Hook F., and its cellular target has not been identified. Here we showed that triptonide effectively inhibited canonical Wnt/β-catenin signaling by targeting a very downstream nuclear component associated with c-termini-transaction domain of β-catenin. Tripterygium wilfordii Hook F., which contains triptonide, has been traditionally used in clinic in China, suggesting that triptonide may have ideal drug properties. Our discovery of triptonide as an effective Wnt inhibitor may represent an important step in identification of bioactive small molecule drugs to stimulate endogenous cardiac progenitor cells for cardiac repair.

Abstract 350: Chromosome Content of Cardiac Progenitor Cells Influence on Regenerative Potential in the Heart

Discovery of endogenous stem cells found within the heart, cardiac progenitor cells (CPC), has prompted intense basic discovery in multiple experimental animal models and clinical trials in heart failure patients. A survey of the literature reveals animal or cellular models exhibiting regenerative properties are also characterized by genome duplication or polyploidization. Our lab recently discovered a fundamental difference between large and small mammalian CPCs through karyotype analysis: rodent (rat, mouse) CPCs possess mononuclear tetraploid (4n) chromosome content, whereas large animal (human, swine) CPCs are mononuclear diploid (2n). This primary distinction between large and small animals prompts provocative questions regarding regenerative potential as well as the translational applicability of regenerative studies performed in rodent models. To expand on this finding, CPCs were isolated from eight human tissue samples (normal and heart failure), two swine strains (Gottingen and Yorkshire) six mice strains (FVB, C57, CAST, SPRET, SAMP6, and SAMR1), and seven rat clonal lines (Sprague Dawley), which confirmed karyotyped ploidy content per species as characterized through flow cytometry and confocal microscopy techniques. The large (human, swine) and small (rat, mouse) mammal CPCs maintained stable chromosome content through increased passaging and positive correlations appears between chromosome content and growth rate through increased passages as well as chromosome content and unrestrained growth through increased passages. The ploidy content of endogenous CPCs was investigated in situ with heart tissue sections with comparable ploidy determinations to that of cultured CPCs. Furthermore, the unique tetraploid content of murine CPCs is reinforced by comparison with ckit+ progenitor cells of secondary tissues (intestine and bone marrow) and cardiomyocytes in situ and in vitro that exhibit mostly diploid (2n) chromosome content per nuclei. These studies suggest ploidy may play a significant role in the biological capacity of the CPC and the endogenous stem cell pool may influence the regenerative capacity of the heart. Future directions will focus on defining the advantage of polyploid content in mediating regeneration.

Cardiac progenitor cells for heart repair.

Stem cell therapy is being investigated as an innovative and promising strategy to restore cardiac function in patients with heart failure. Several stem cell populations, including adult (multipotent) stem cells from developed organs and tissues, have been tested for cardiac repair with encouraging clinical and pre-clinical results. The heart has been traditionally considered a post-mitotic organ, however, this view has recently changed with the identification of stem/progenitor cells residing within the adult heart. Given their cardiac developmental origins, these endogenous cardiac progenitor cells (CPCs) may represent better candidates for cardiac cell therapy compared with stem cells from other organs such as the bone marrow and adipose tissue. This brief review will outline current research into CPC populations and their cardiac repair/regenerative potential.

In This Issue

HomeCirculation ResearchVol. 118, No. 7In This Issue Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBIn This Issue Originally published1 Apr 2016https://doi.org/10.1161/RES.0000000000000102Circulation Research. 2016;118:1041Glut1, Myelopoiesis, and Atherosclerosis (p 1062)Download figureDownload PowerPointInhibiting extramedullary hematopoietic activity may halt progression of atherosclerosis, say Sarrazy et al.To proliferate and differentiate hematopoietic stem and progenitor cells (HSPCs) in the bone marrow increase their glucose uptake to meet the rising metabolic demand. Indeed PET-CT imaging can identify increased metabolic activity in HSPCs by the uptake of glucose (or rather its radiolabeled analogue) during bone marrow and extramedullary hematopoietic activity. Cell proliferation dependent glucose uptake has been observed in atherosclerotic plaques as well as the spleen. Overproduction of hematopoietic cells, including neutrophils and monocytes, could both contribute to atherosclerosis onset and exacerbate plaque growth. To study extramedullary hematopoiesis more closely, Sarrazy and colleagues examined atherosclerosis-prone mice. They found that glucose uptake was increased in the aortic arch, spleens and bone marrow of these animals, which correlated with increased expression of the glucose transporter Glut1 in their HSPCs. Furthermore, suppression of Glut1 activity reduced not only HSPC proliferation and differentiation—specifically myelopoiesis—but also the progression of atherosclerosis. The authors propose that preventing glucose uptake by HSPCs with an appropriately targeted Glut1 inhibitor could be a potential treatment strategy for atherosclerosis.Long-Term Effects of Cardiac Progenitor Cells (p 1091)Download figureDownload PowerPointThe benefits of cardiac progenitor cell therapy lasts at least a year in rats, report Tang et al.Several studies show that the therapeutic use of cardiac progenitor cells (CPCs) can promote repair and improve function of damaged hearts in animals after myocardial infarction. However, long-term effects of CPC therapy remain unknown as most studies have examined the potential benefits for only four-to-six weeks. Therefore, Tang and colleagues studied the outcome of CPC therapy for myocardial infarction in rats, one year after CPC transplant. They found that while the overall survival rate was no different between control and CPC-treated animals, the treated group exhibited less hypertrophy, smaller scars, increased left ventricle wall thickness, and evidence of less fibrosis. Significantly, CPC treatment did not result in tumor formation. The team used male CPCs and female recipients to enable identification of the cells—by the Y chromosome—and this approach revealed that while a small number of the cells were still present even after 1 year, the transplanted cells had not differentiated into mature cardiomyocytes, although increased proliferation of endogenous CPCs was observed. Together these results indicate that CPC therapy is safe, and that it provides long-term benefits, and induces endogenous CPCs to proliferate and promote repair. The study paves the way for long-term studies evaluating the clinical efficacy of stem cells, say the authors.GRK2 and Prognosis in Heart Failure (p 1116)Download figureDownload PowerPointRengo et al assess the usefulness of a possible new biomarker for heart failure.Heart failure (HF) is a leading cause of death worldwide, and about five million people in the United States suffer from the condition. A major characteristic of HF is chronic sympathetic nervous system (SNS) hyperactivity, which while compensatory in the initial stages of disease becomes deleterious in the long run. Indeed, chronic SNS hyperactivity boosts cardiac levels of G-protein coupled receptor (GPCR) kinase 2 (GRK2), which in turn leads to dysfunctional β-adrenergic signaling—a key factor contributing to the progress of HF pathology. Importantly, the levels of GRK2 in peripheral lymphocytes correlate with those in the failing heart. Rengo and colleagues studied levels of lymphocyte GRK2 in 257 HF patients and found that they accurately reflect disease severity. Patients at low risk of death has lower levels of GRK2, while high levels of protein were identified in high risk individuals. Currently, left ventricle ejection fraction and levels of NT-proBNP are used as biomarkers for HF; however, the team suggests that GRK2 could offer independent and additional prognostic value over and above current approaches. The authors therefore suggest that further larger-scale clinical investigations are warranted to assess the value of the prognostic value of GRK2 in heart failure patients. Previous Back to top Next FiguresReferencesRelatedDetails April 1, 2016Vol 118, Issue 7 Advertisement Article InformationMetrics © 2016 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000102 Originally publishedApril 1, 2016 PDF download Advertisement

Nuclear Calcium/Calmodulin-dependent Protein Kinase II Signaling Enhances Cardiac Progenitor Cell Survival and Cardiac Lineage Commitment

Ca(2+)/Calmodulin-dependent protein kinase II (CaMKII) signaling in the heart regulates cardiomyocyte contractility and growth in response to elevated intracellular Ca(2+). The δB isoform of CaMKII is the predominant nuclear splice variant in the adult heart and regulates cardiomyocyte hypertrophic gene expression by signaling to the histone deacetylase HDAC4. However, the role of CaMKIIδ in cardiac progenitor cells (CPCs) has not been previously explored. During post-natal growth endogenous CPCs display primarily cytosolic CaMKIIδ, which localizes to the nuclear compartment of CPCs after myocardial infarction injury. CPCs undergoing early differentiation in vitro increase levels of CaMKIIδB in the nuclear compartment where the kinase may contribute to the regulation of CPC commitment. CPCs modified with lentiviral-based constructs to overexpress CaMKIIδB (CPCeδB) have reduced proliferative rate compared with CPCs expressing eGFP alone (CPCe). Additionally, stable expression of CaMKIIδB promotes distinct morphological changes such as increased cell surface area and length of cells compared with CPCe. CPCeδB are resistant to oxidative stress induced by hydrogen peroxide (H2O2) relative to CPCe, whereas knockdown of CaMKIIδB resulted in an up-regulation of cell death and cellular senescence markers compared with scrambled treated controls. Dexamethasone (Dex) treatment increased mRNA and protein expression of cardiomyogenic markers cardiac troponin T and α-smooth muscle actin in CPCeδB compared with CPCe, suggesting increased differentiation. Therefore, CaMKIIδB may serve as a novel modulatory protein to enhance CPC survival and commitment into the cardiac and smooth muscle lineages.

Abstract 17: Nuclear Calcium/Calmodulin-dependent Protein Kinase II Signaling Enhances Cardiac Progenitor Cell Survival and Cardiac Lineage Commitment

Ca2+/Calmodulin-dependent protein kinase II (CaMKII) signaling in the heart regulates cardiomyocyte contractility and growth in response to elevated intracellular Ca2+. The δB isoform of CaMKII is the predominant nuclear splice variant in the adult heart and regulates cardiomyocyte hypertrophic gene expression by signaling to the histone deacetylase HDAC4. However, the role of CaMKIIδ in cardiac progenitor cells (CPCs) has not been explored. During developmental growth endogenous CPCs display primarily cytosolic CaMKIIδ, which localizes to the nuclear compartment of CPCs after myocardial infarction injury. CPCs undergoing early differentiation in vitro increase levels of CaMKIIδB in the nuclear compartment where the kinase may contribute to the regulation of CPC commitment. CPCs modified with an established lentiviral based constructs to overexpress CaMKIIδB (CPCeδB) have reduced proliferative rate compared to lentiviral transduction of CPCs with eGFP alone (CPCe). Additionally, stable expression of CaMKIIδB promotes distinct morphological changes such as increased cell surface area and increased length of cells compared to CPCe. CPCeδB are resistant to oxidative stress induced by H2O2 relative to CPCe, whereas a knockdown of CaMKIIδB using small hairpin RNA resulted in an up regulation of cell death compared to scrambled treated controls. Dexamethasone treatment to promote cardiac differentiation increased cardiomyogenic markers cardiac troponin T and α-smooth muscle actin measured by RT-PCR and immunoblot analyses in CPCeδB compared to control CPCe. Therefore, CaMKIIδB may serve as a novel modulatory protein to enhance CPC survival and commitment into the cardiac and smooth muscle lineage.

Recent developments in cardiovascular stem cells.

Heart failure, a common consequence of ischemic heart disease, is a major cause of morbidity and mortality in the world.1–3 Pharmacological treatment with β-blockers and inhibitors of the renin–angiotensin–aldosterone system has improved the clinical outcomes in patients with heart failure.4–7 Likewise, mechanical unloading with left ventricular assist devices and resynchronization therapy have led to partial reversal of cardiac structural and molecular remodeling and symptomatic improvement.8–10 Despite these remarkable advances, however, mortality and morbidity of patients with heart failure, with or without reduced ejection fraction remains high.1,2 Moreover, heart transplantation, while an effective option, is available only for a selected number of patients and is not without considerable negative consequences.11 Furthermore, gene therapy still remains in early investigational stages and not yet ready for clinical applications.12 The high residual mortality and morbidity of patients with heart failure might be inherent to the shortcomings of the current therapeutic approaches, as none directly targets the underlying causal problem in heart failure, that is, loss of or intrinsically dysfunctional myocytes. Consequently, novel therapeutic approaches are necessary to further improve the clinical outcomes in patients with heart failure. The heart is considered, by and large, a terminally differentiated organ with a limited intrinsic regenerative capacity that alone is insufficient to compensate for the pathological loss of cardiac myocytes during the postnatal period.13–15 The discovery of cardiac progenitor cells (CPCs) in the heart more than a decade ago along with the recent data showing that the existing myocytes undergo a gradual turnover have raised the potentials for regenerative cardiac repair.16,17 Likewise, the discovery of mesenchymal stem cells (MSCs), which was thought to have the potential to differentiate to cardiac myocytes, but yet to proven, or enhance …

Brain natriuretic peptide is able to stimulate cardiac progenitor cell proliferation and differentiation in murine hearts after birth.

Brain natriuretic peptide (BNP) contributes to heart formation during embryogenesis. After birth, despite a high number of studies aimed at understanding by which mechanism(s) BNP reduces myocardial ischemic injury in animal models, the actual role of this peptide in the heart remains elusive. In this study, we asked whether BNP treatment could modulate the proliferation of endogenous cardiac progenitor cells (CPCs) and/or their differentiation into cardiomyocytes. CPCs expressed the NPR-A and NPR-B receptors in neonatal and adult hearts, suggesting their ability to respond to BNP stimulation. BNP injection into neonatal and adult unmanipulated mice increased the number of newly formed cardiomyocytes (neonatal: +23 %, p = 0.009 and adult: +68 %, p = 0.0005) and the number of proliferating CPCs (neonatal: +142 %, p = 0.002 and adult: +134 %, p = 0.04). In vitro, BNP stimulated CPC proliferation via NPR-A and CPC differentiation into cardiomyocytes via NPR-B. Finally, as BNP might be used as a therapeutic agent, we injected BNP into mice undergoing myocardial infarction. In pathological conditions, BNP treatment was cardioprotective by increasing heart contractility and reducing cardiac remodelling. At the cellular level, BNP stimulates CPC proliferation in the non-infarcted area of the infarcted hearts. In the infarcted area, BNP modulates the fate of the endogenous CPCs but also of the infiltrating CD45(+) cells. These results support for the first time a key role for BNP in controlling the progenitor cell proliferation and differentiation after birth. The administration of BNP might, therefore, be a useful component of therapeutic approaches aimed at inducing heart regeneration.

Abstract 19616: Ischemic Stress Reduces Cardiac Progenitor Cell Self-renewal Through Gsk3β Activation and Reduction in Cmyc Stability

Introduction: The adult heart harbors a population of resident stem cells capable of self-renewal and differentiation into cardiac, smooth muscle and endothelial lineages. Cardiac progenitor cells (CPCs) have been shown to promote cardiac regeneration and improve heart function. However, despite their potential use in cardiac repair, evidence suggests that resident CPCs regenerative capacity is limited in conditions of severe hypoxia such as ischemic cardiomyopathy. Elucidation of the mechanisms involved in CPC protection against hypoxic stress is essential to maximize their cardioprotective and therapeutic potential. Methods and Results: We investigated the growth pattern of murine CPCs under normoxia (21% oxygen) and hypoxia (0.5% oxygen) conditions along a period of 96 hours. We found that CPC proliferation in hypoxia was significantly reduced by 40% after 72-96 hours (p&lt;0.05, n=4-7), without evidence of increase in cell death. CPC proliferation was accompanied with a time-dependent increase in senescence-associated β-galactosidase activity by 30% in hypoxia relative to normoxia control (p&lt;0.02, n=7). Western blot analysis of CPCs grown under normoxia and hypoxia revealed a time-dependent decrease in the expression of the transcription factor cMyc and its downstream target Bmi-1 by 40% and 30% (p&lt;0.05, n=4-5) relative to normoxia control, respectively. Interestingly, reduction in cMyc protein expression was not associated with a decrease in cMyc transcripts, suggesting that cMyc protein degradation is activated under hypoxia. Treatment of CPCs with cycloheximide to block protein synthesis showed that the rate of cMyc degradation under hypoxia is increased by 30% (p&lt;0.05, n=5) relative to normoxia. GSK3β plays a central role in the control of cMyc stability. Growth of CPCs in the presence of GSK3β inhibitor rescued proliferation in hypoxia by approximately 40% (n=4). Conclusions: Our results suggest that hypoxic stress reduces CPC self-renewal potential by inducing CPC senescence through a mechanism that involves decrease of cMyc stability through GSK3β activation. Modulation of GSK3β activity may be used as a therapeutic approach for protection of endogenous CPCs self-renewal potential under ischemic conditions.