Cardiac Repair Capacity Research Articles

Thrombospondin 1 and Reelin act through Vldlr to regulate cardiac growth and repair.

Adult mammalian cardiomyocytes have minimal cell cycle capacity, which leads to poor regeneration after cardiac injury such as myocardial infarction. Many positive regulators of cardiomyocyte cell cycle and cardioprotective signals have been identified, but extracellular signals that suppress cardiomyocyte proliferation are poorly understood. We profiled receptors enriched in postnatal cardiomyocytes, and found that very-low-density-lipoprotein receptor (Vldlr) inhibits neonatal cardiomyocyte cell cycle. Paradoxically, Reelin, the well-known Vldlr ligand, expressed in cardiac Schwann cells and lymphatic endothelial cells, promotes neonatal cardiomyocyte proliferation. Thrombospondin1 (TSP-1), another ligand of Vldlr highly expressed in adult heart, was then found to inhibit cardiomyocyte proliferation through Vldlr, and may contribute to Vldlr's overall repression on proliferation. Mechanistically, Rac1 and subsequent Yap phosphorylation and nucleus translocation mediate the regulation of the cardiomyocyte cell cycle by TSP-1/Reelin-Vldlr signaling. Importantly, Reln mutant neonatal mice displayed impaired cardiomyocyte proliferation and cardiac regeneration after apical resection, while cardiac-specific Thbs1 deletion and cardiomyocyte-specific Vldlr deletion promote cardiomyocyte proliferation and are cardioprotective after myocardial infarction. Our results identified a novel role of Vldlr in consolidating extracellular signals to regulate cardiomyocyte cell cycle activity and survival, and the overall suppressive TSP-1-Vldlr signal may contribute to the poor cardiac repair capacity of adult mammals.

Differential gene responses 3 days following infarction in the fetal and adolescent sheep heart.

There are critical molecular mechanisms that can be activated to induce myocardial repair, and in humans this is most efficient during fetal development. The timing of heart development in relation to birth and the size/electrophysiology of the heart are similar in humans and sheep, providing a model to investigate the repair capacity of the mammalian heart and how this can be applied to adult heart repair. Myocardial infarction was induced by ligation of the left anterior descending coronary artery in fetal (105 days gestation when cardiomyocytes are proliferative) and adolescent sheep (6 mo of age when all cardiomyocytes have switched to an adult phenotype). An ovine gene microarray was used to compare gene expression in sham and infarcted (remote, border and infarct areas) cardiac tissue from fetal and adolescent hearts. The gene response to myocardial infarction was less pronounced in fetal compared with adolescent sheep hearts and there were unique gene responses at each age. There were also region-specific changes in gene expression between each age, in the infarct tissue, tissue bordering the infarct, and tissue remote from the infarction. In total, there were 880 genes that responded to MI uniquely in the adolescent samples compared with 170 genes in the fetal response, as well as 742 overlap genes that showed concordant direction of change responses to infarction at both ages. In response to myocardial infarction, there were specific changes in genes within pathways of mitochondrial oxidation, muscle contraction, and hematopoietic cell lineages, suggesting that the control of energy utilization and immune function are critical for effective heart repair. The more restricted gene response in the fetus may be an important factor in its enhanced capacity for cardiac repair.

Abstract 132: Imaging-Based Assay for Screening of Cell Cycle Modifying Substances in Postnatal Cardiomyocytes

Cardiovascular diseases are the main cause of death in the industrial world. Especially large myocardial infarctions and the potentially resulting left ventricular dysfunction are chronical diseases with a poor prognosis. Due to limited endogenous cardiac repair capacity, we are investigating the regulation of the cell cycle in cardiomyocytes (CMs) as a potential treatment strategy. We hypothesize that controlled induction of cardiomyocyte proliferation will lead to an increase in muscle mass and improvement of cardiac function. We are using CMs from double transgenic αMHC-H2B-mCherry/CAG-eGFP-anillin mice for the analysis of postnatal CM development. This double transgenic system enables us to unequivocally identify CM nuclei as well as cell cycle variations like acytokinetic mitosis (mitosis without cytokinesis), resulting in binucleated CMs, and endoreplication (mitosis without cytokinesis and karyokinesis), leading to polyploid CMs. With the goal of inducing authentic cell division, we are screening for pro-proliferative substances in P1 and P6 postnatal CMs of αMHC-H2B-mCherry/CAG-eGFP-anillin mice. Here we will present the detailed analysis of several screening hits demonstrating an increase in cell cycle activity leading mainly to binucleation but only to limited cell division in CMs.

P945PCSK9 deficiency is not associated with impaired cardiac repair capacity early after myocardial infarction

P945PCSK9 deficiency is not associated with impaired cardiac repair capacity early after myocardial infarction

Collagen scaffold enhances the regenerative properties of mesenchymal stromal cells.

MSCs are widely applied to regenerate heart tissue in myocardial diseases but when grown in standard two-dimensional (2D) cultures exhibit limited potential for cardiac repair and develop fibrogenic features with increasing culture time. MSCs can undergo partial cardiomyogenic differentiation, which improves their cardiac repair capacity. When applied to collagen patches they may improve cardiac tissue regeneration but the mechanisms remain elusive. Here, we investigated the regenerative properties of MSCs grown in a collagen scaffold as a three-dimensional (3D) culture system, and performed functional analysis using an engineered heart tissue (EHT) model. We showed that the expression of cardiomyocyte-specific proteins by MSCs co-cultured with rat neonatal cardiomyocytes was increased in collagen patches versus conventional cultures. MSCs in 3D collagen patches were less fibrogenic, secreted more cardiotrophic factors, retained anti-apoptotic and immunomodulatory function, and responded less to TLR4 ligand lipopolysaccharide (LPS) stimulation. EHT analysis showed no effects by MSCs on cardiomyocyte function, whereas control dermal fibroblasts abrogated the beating of cardiac tissue constructs. We conclude that 3D collagen scaffold improves the cardioprotective effects of MSCs by enhancing the production of trophic factors and modifying their immune modulatory and fibrogenic phenotype. The improvement in myocardial function by MSCs after acquisition of a partial cardiac cell-like phenotype is not due to enhanced MSC contractility. A better understanding of the mechanisms of MSC-mediated tissue repair will help to further enhance the therapeutic potency of MSCs.

Abstract 15044: Cardiac Explant-derived Cells From Aged Donors With Ischemic Cardiomyopathy Demonstrate Impaired Cell Survival and Therapeutic Regeneration

Background: While cardiac stem cells are emerging as a promising therapy for ischemic cardiomyopathy (ICM), the influence of advanced donor age and ICM on the regenerative performance of these cells are unknown. Hypothesis: Advanced donor age and a history of ICM combine to limit the regenerative performance of explant-derived cells (EDCs) through altered cell functions which reflects fundamental changes in the molecular signature of aged ischemic EDCs. Methods and Results: EDCs were cultured from myocardial biopsies of young (8 weeks; n=40) and old (52 weeks; n=36) C57/BL6 mice 4 weeks after randomization to sham or experimental myocardial infraction (MI; ejection fraction 28±2%, p=0.24 effect of donor age). Although advanced donor age was associated with reduced telomere length, aging alone had negligible effects on EDCs. In contrast, EDCs sourced from aged ICM donors showed reduced cardiomyogenic differentiation (i.e., greater propensity to adopt an endothelial or smooth muscle lineage), cytokine production (reduced angiogenin, angiopoietin-1 and VEGF), pro-angiogenic capacity (1.2±0.03 fold less tubules formed; p=0.02), reactive oxygen species metabolism (1.8±0.3 fold greater reactive oxygen species, p=0.04; 1.9±0.1 fold less superoxide dismutase activity, p<0.01; 2.4±0.3 fold less glutathione peroxidase activity, p<0.01), and apoptosis resistance (1.5±0.1 fold greater; p=0.02) as compared to young ICM EDCs. With the exception of 1.6±0.1 fold greater production of IL-6 (p=0.04), ICM did not influence young EDCs. Transplantation of EDCs from aged ICM donors into aged recipients 1 week after MI provided lesser degrees of cell-mediated cardiac repair (3±1 vs. 8±1% change in LVEF from baseline to 21 days post CSC transplant after transplant of young ICM EDCs; p<0.01). Finally, transcriptome comparison of ICM EDCs showed aging promoted the differential expression of 145 genes (≥2 fold change; false discovery rate ≤0.05) which modify many of the pathways responsible for effective cell cycle control and DNA damage/repair. Conclusions: EDCs from aged donors with a history of ICM demonstrate an attenuated capacity for cardiac repair that reflects differential expression of transcripts suggestive of impaired cell cycle control and repair.

THE MOLECULAR SIGNATURE OF CSCS SOURCED FROM AGED DONORS WITH ISCHEMIC CARDIOMYOPATHY IMPAIRS CELL CYCLE CONTROL AND REPAIR

Aging reduces the ability of organs to maintain homeostasis and respond to stressors. Previously, we have shown that aging increases the production of pro-inflammatory cytokines by ex vivo proliferated cardiac stem cells (CSCs) while ischemic aged CSCs have shorter telomere lengths, higher levels of oxidative stress and are less capable of repairing injured myocardium after transplantation. In this study, we explore the fundamental mechanisms underlying these changes. Young (YM; 8 wks) and old (OM; 52 wks) C57/BL6 mice were randomized to receive no intervention (n=20 YM and n=19 OM) or surgical ligation of the left anterior descending artery (n=20 YM and n=17 OM). Following establishment of ischemic cardiomyopathy (ICM) (+4 week ejection fraction: 27±2% and 30±2% for OM and YM, respectively, p=0.24), CSCs were cultured from plated ICM or age matched myocardial biopsies. After exposure to low serum hypoxic (1% oxygen) stress conditions designed to mirror the remodeled ICM heart, flow cytometry demonstrated that while aging alone had little negligible effects on CSC viability, aging and a history of ICM combined to increase the number of apoptotic CSCs by 1.5±0.2 fold (p=0.01). Consistent with these findings, microarray profiling revealed that aging alone prompted the differential expression of 245 genes (≥2 fold change; false discovery rate (FDR) ≤0.05) that resulted in up-regulation of pro-inflammatory pathways including NF-КB, TREM1, Toll-like receptor and death receptor signaling. A history of ICM resulted in the differential expression of 924 and 773 genes in OM and YM donors, respectively (≥2 fold change; FDR≤0.05). ICM resulted in CSCs sourced from YM donors up-regulating transcripts responsible for cell migration, proliferation, survival within the Protein Kinase A, NRF2-mediated oxidative stress response and PPAR signalling pathways. In contrast, a history of ICM in OM donors increased stimulation within pathways responsible for IL-6, NF-КB, TNFR2, TREM1 and p38 MAPK signaling. Most interestingly, aging alone promoted the differential expression of 145 genes in ICM CSCs (≥2 fold change; FDR≤0.05) which modify many of the pathways responsible for effective cell cycle control and DNA damage/repair. Although advanced donor age biases ex vivo proliferated CSCs to adopt a pro-inflammatory molecular signature, a history of ICM promotes the expression of pro-survival pro-cardiogenic transcripts- suggestive of a tendency towards enhanced cardiac repair. In contrast, CSCs from aged donors with a history of ICM exhibit transcripts underlying an attenuated capacity for cardiac repair through impaired cell cycle control and repair.

Loss of AngiomiR-126 and 130a in Angiogenic Early Outgrowth Cells From Patients With Chronic Heart Failure

Background— MicroRNAs are key regulators of angiogenic processes. Administration of angiogenic early outgrowth cells (EOCs) or CD34 + cells has been suggested to improve cardiac function after ischemic injury, in particular by promoting neovascularization. The present study therefore examines regulation of angiomiRs, microRNAs involved in angiogenesis, in angiogenic EOCs and circulating CD34 + cells from patients with chronic heart failure (CHF) and the role for their cardiac repair capacity. Methods and Results— Angiogenic EOCs and CD34 + cells were isolated from patients with CHF caused by ischemic cardiomyopathy (n=45) and healthy subjects (n=35). In flow cytometry analyses, angiogenic EOCs were largely myeloid and positive for alternatively activated M2 macrophage markers. In vivo cardiac neovascularization and functional repair capacity were examined after transplantation into nude mice with myocardial infarction. Cardiac transplantation of angiogenic EOCs from healthy subjects markedly increased neovascularization and improved cardiac function, whereas no such effect was observed after transplantation of angiogenic EOCs from patients with CHF. Real-time polymerase chain reaction analysis of 14 candidate angiomiRs, expressed in angiogenic EOCs, revealed a pronounced loss of angiomiR-126 and -130a in angiogenic EOCs from patients with CHF that was also observed in circulating CD34 + cells. Anti–miR-126 transfection markedly impaired the capacity of angiogenic EOCs from healthy subjects to improve cardiac function. miR-126 mimic transfection increased the capacity of angiogenic EOCs from patients with CHF to improve cardiac neovascularization and function. Conclusions— The present study reveals a loss of angiomiR-126 and -130a in angiogenic EOCs and circulating CD34 + cells from patients with CHF. Reduced miR-126 expression was identified as a novel mechanism limiting their capacity to improve cardiac neovascularization and function that can be targeted by miR-126 mimic transfection.

Circulation: Clinical Summaries

<i>Circulation:</i> Clinical Summaries

Hepatocyte growth factor mobilizes non-bone marrow-derived circulating mesoangioblasts

The identification of factors that mobilize subsets of endogenous progenitor cells may provide new therapeutic tools to enhance the repair of ischaemic tissue. We previously identified circulating mesenchymal cells that co-express endothelial markers (so-called circulating mesoangioblasts, cMABs) in children undergoing heart surgery with cardiopulmonary bypass (CPB). However, the mechanisms by which these cells are mobilized and their origin is unclear. Circulating CD73(+)CD45(-)KDR(+) cMABs were analysed in adults undergoing heart surgery with (n = 21) or without CPB (n = 8). During surgery with CPB, cMABs are mobilized with a maximal response at the end of the operation. In contrast, off-pump heart surgery does not stimulate cMAB mobilization, indicating that the stress mediated by CPB induces the mobilization of cMAB. Circulating mesoangioblasts were enriched in blood obtained from the coronary sinus. Histologically, CD73(+) cells were detected around vessels in the heart, indicating that the heart is one of the niches of cMABs. Consistently, studies in gender mismatched bone marrow transplanted patients demonstrated that cMABs did not originate from the bone marrow. Cytokine profiling of serum samples revealed that hepatocyte growth factor (HGF) was profoundly increased at the time point of maximal mobilization of cMABs. Hepatocyte growth factor stimulated the migration of cMABs. Importantly, injection of recombinant HGF increased cMABs in rats. Hepatocyte growth factor induces mobilization of non-haematopoietic progenitor cells with a cardiac repair capacity. This newly identified function together with the known pleiotrophic effects of HGF makes HGF an attractive therapeutic option for the treatment of ischaemic heart disease.

Bone Marrow Mesenchymal Stem Cells Stimulate Cardiac Stem Cell Proliferation and Differentiation

The regenerative potential of the heart is insufficient to fully restore functioning myocardium after injury, motivating the quest for a cell-based replacement strategy. Bone marrow-derived mesenchymal stem cells (MSCs) have the capacity for cardiac repair that appears to exceed their capacity for differentiation into cardiac myocytes. Here, we test the hypothesis that bone marrow derived MSCs stimulate the proliferation and differentiation of endogenous cardiac stem cells (CSCs) as part of their regenerative repertoire. Female Yorkshire pigs (n=31) underwent experimental myocardial infarction (MI), and 3 days later, received transendocardial injections of allogeneic male bone marrow-derived MSCs, MSC concentrated conditioned medium (CCM), or placebo (Plasmalyte). A no-injection control group was also studied. MSCs engrafted and differentiated into cardiomyocytes and vascular structures. In addition, endogenous c-kit(+) CSCs increased 20-fold in MSC-treated animals versus controls (P<0.001), there was a 6-fold increase in GATA-4(+) CSCs in MSC versus control (P<0.001), and mitotic myocytes increased 4-fold (P=0.005). Porcine endomyocardial biopsies were harvested and plated as organotypic cultures in the presence or absence of MSC feeder layers. In vitro, MSCs stimulated c-kit(+) CSCs proliferation into enriched populations of adult cardioblasts that expressed Nkx2-5 and troponin I. MSCs stimulate host CSCs, a new mechanism of action underlying successful cell-based therapeutics.

Increased Cardiac Myocyte Progenitors in Failing Human Hearts

Increasing evidence, derived mainly from animal models, supports the existence of endogenous cardiac renewal and repair mechanisms in adult mammalian hearts that could contribute to normal homeostasis and the responses to pathological insults. Translating these results, we isolated small c-kit+ cells from 36 of 37 human hearts using primary cell isolation techniques and magnetic cell sorting techniques. The abundance of these cardiac progenitor cells was increased nearly 4-fold in patients with heart failure requiring transplantation compared with nonfailing controls. Polychromatic flow cytometry of primary cell isolates (<30 microm) without antecedent c-kit enrichment confirmed the increased abundance of c-kit+ cells in failing hearts and demonstrated frequent coexpression of CD45 in these cells. Immunocytochemical characterization of freshly isolated, c-kit-enriched human cardiac progenitor cells confirmed frequent coexpression of c-kit and CD45. Primary cardiac progenitor cells formed new human cardiac myocytes at a relatively high frequency after coculture with neonatal rat ventricular myocytes. These contracting new cardiac myocytes exhibited an immature phenotype and frequent electric coupling with the rat myocytes that induced their myogenic differentiation. Despite the increased abundance and cardiac myogenic capacity of cardiac progenitor cells in failing human hearts, the need to replace these organs via transplantation implies that adverse features of the local myocardial environment overwhelm endogenous cardiac repair capacity. Developing strategies to improve the success of endogenous cardiac regenerative processes may permit therapeutic myocardial repair without cell delivery per se.