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Following the publication of this paper, it was drawn to the Editors' attention by a concerned reader that the immunofluorescence data shown in Fig. 2G, the mitochondria‑ and lysosome‑stained images in Fig. 3C, the JC‑1 staining images in Fig. 4C and the immunofluorescence data in Fig. 5G were strikingly similar to data appearing in different form in other articles written by different authors at different research institutes that had either already been published elsewhere prior to the submission of this paper to Molecular Medicine Reports , or were under consideration for publication at around the same time. In view of the fact that certain of the abovementioned data had already apparently been published previously, the Editor of Molecular Medicine Reports has decided that this paper should be retracted from the Journal. After having been in contact with the authors, they agreed with the decision to retract the paper. The Editor apologizes to the readership for any inconvenience caused. [Molecular Medicine Reports 17: 3722‑3734, 2018; DOI: 10.3892/mmr.2018.8371].

Many advances have been made in moving towards repairing the infarcted ischemic heart, but clinical translation remains challenging. It is unlikely that the solution of repairing the heart lies in a single factor targeting a single process. The main objective of the research in this thesis is to explore methods to protect the heart from ischemic and I/R injury and stimulate the innate reparative capacity of the heart to renew damaged myocardium. To provide scope to achieve these complex tasks, we subdivided the research into answering three consecutive questions: 1. How can we protect human cardiac cells from I/R injury? 2. How can we stimulate human cardiomyocytes to generate new functional cardiomyocytes to repair injured tissue? 3. If we have a protective and reparative therapy available, how can we deliver it safely and effectively to the right location in the heart? Chapter 2 focusses on the first step towards cardiac repair: limiting the damage inflicted during an ischemic event and reperfusion, the current golden standard of therapy. In this chapter we introduce a novel therapeutic factor that efficiently targets necroptotic cell death, a major form of cell death determining I/R injury. In Chapter 3, we develop a hiPSC-cardiomyocyte based platform to model human IHD. To do this, we assessed the role of metabolic maturation on susceptibility of hiPSC-cardiomyocytes to hypoxic damage. Chapter 4 discusses the potential of stimulating cardiomyocyte proliferation to regenerate the heart by focussing on the role of miRNA-128 in cardiac regeneration. Chapter 5 introduces the glycoprotein Follistatin-like 1 as a potential therapeutic tool to prevent cardiomyocyte cell death, increase vascularisation in the infarcted area and stimulate cardiomyocyte renewal. In Chapter 6, we used our model developed in chapter 3 to test the potential of Follistatin-like 1 as a therapeutic factor to treat human IHD. In Chapter 7, we discuss the role of non-coding RNAs in cardiac regeneration and vascularisation and how to provide localized and sustained delivery of non-coding RNAs to the infarcted region. Chapter 8 describes a new method we developed to assess the retention of injected hydrogels after cardiac injection using real-time tracking with radioactively labelled hydrogel subunits.Chapter 9 provides a summary and general discussion of the work presented in this thesis and discusses future directions.

Infarct healing is dependent on recruitment of inflammatory leukocytes and subsequent activation of myofibroblasts (MF) and neovessel formation, ultimately resulting in formation of a highly vascularized collagen-enriched scar. Though the heart has an abundant population of periendothelial pericytes, its role in wound healing upon myocardial infarction (MI) has not been studied. We hypothesized that in the infarcted myocardium, pericytes may become activated, contributing to inflammatory, fibrotic and angiogenic responses. We used pericyte/fibroblast reporter mice (NG2 DsRed ;PDGFRα GFP ), lineage tracing studies and in vitro approaches to study the fate and role of pericytes in the infarcted myocardium. In normal hearts, NG2+/PDGFRα- pericytes and PDGFRα+/NG2- fibroblasts had distinct transcriptomic profiles. Pericytes expressed mural genes like Acta2 , Pdgfrb and low amounts of extracellular matrix (ECM) genes, whereas fibroblasts synthesized collagens, Timp2/3 and matricellular genes. 7 days post-MI, expansion of the NG2+ population in the infarct zone was associated with emergence of non-mural NG2+/αSMA+ cells with MF characteristics. FACS-sorted NG2+/PDGFRα- cells from 7-day infarcts expressed higher levels of collagens when compared to NG2+/PDGFRα- cells from normal hearts. Infarct pericytes had high integrin and MMP14 expression, reflecting an activated migratory phenotype. Lineage tracing using NG2CreER TM ;Rosa tdTomato ;PDGFRα GFP mice showed that 5.7%±1.04 of PDGFRα+ fibroblasts and 10.49%±2.73 of infarct MFs were derived from NG2+ lineage. Pericyte-derived fibroblasts exhibited higher ECM gene synthesis, in comparison to fibroblasts from non-pericyte origin, while pericyte-derived mural cells showed accentuated inflammatory cytokine gene expression. Immunostaining showed pericytes actively contribute to vascular maturation, forming a mural cell coat enwrapping infarct neovessels. In vitro, TGFβ induced integrins, collagens and MMPs in human pericytes, similar to the changes observed in infarct pericytes. Taken together, our evidences show that after MI, pericytes become activated and contribute to repair by undergoing conversion to a subset of myofibroblasts and by coating infarct neovessels.

Abundant macrophages infiltrate the infarcted heart and play a critical role in repair, remodeling and fibrosis. Macrophages sense changes in the extracellular matrix (ECM) environment through Integrins, thus activating signaling pathways that regulate their function. Analysis of our RNA sequencing data identified integrin α5 (Itgα5) as one of the most upregulated integrin genes in infarct macrophages. Accordingly, we hypothesized that integrin α5 signaling in infarct macrophages transduces ECM-derived signals, regulating responses critical for repair and remodeling of the infarcted heart.In order to study the role of macrophage α5 integrin in the infarcted heart, we generated 2 lines of macrophage-specific α5 integrin KO mice: myeloid cell-specific KOs (Myα5KO, using the lysozyme-M Cre driver) and inducible macrophage-specific α5 KOs (iMaα5KO, using CX3CR1 CreER ). 28 days after infarction, Myα5KO mice had accentuated adverse remodeling, evidenced by increased LVEDV and LVESV, a trend towards reduced ejection fraction. Adverse remodeling in Mya5KO was associated with a marked reduction in microvascular density in the infarct and in the border zone. iMaα5KO mice also exhibited worse remodeling and impaired infarct angiogenesis. A PCR array in infarct macrophages showed that α5 integrin loss was associated with markedly reduced transcription of VEGF-A, and lower levels of the angiogenic CXC chemokines CXCL1 and CXCL2. RNAseq followed by bioinformatic analysis predicted that that Nrf2, Erk5, HIF1a, p38 MAPK, VEGF, RhoA, and PI3K/Akt pathways were inhibited in the absence of α5 signaling in vivo (in infarct macrophages) and in vitro (in bone marrow macrophages undergoing α5 antibody neutralization experiments).In conclusion, α5 integrin promotes an angiogenic VEGF-expressing phenotype in infarct macrophages, protecting the infarcted heart from adverse remodeling. The angiogenic effects of α5 integrin in macrophages may have important therapeutic implications for heart failure patients.

Direct remuscularization approaches to cell-based heart repair seek to restore ventricular contractility following myocardial infarction (MI) by introducing new cardiomyocytes (CMs) to replace lost or injured ones. However, despite promising improvements in cardiac function, high incidences of ventricular arrhythmias have been observed in animal models of MI injected with pluripotent stem cell-derived cardiomyocytes (PSC-CMs). The mechanisms of arrhythmogenesis remain unclear. Here, we present a comprehensive framework for computational modeling of direct remuscularization approaches to cell therapy. Our multiscale 3D whole-heart modeling framework integrates realistic representations of cell delivery and transdifferentiation therapy modalities as well as representation of spatial distributions of engrafted cells, enabling simulation of clinical therapy and the prediction of emergent electrophysiological behavior and arrhythmogenensis. We employ this framework to explore how varying parameters of cell delivery and transdifferentiation could result in three mechanisms of arrhythmogenesis: focal ectopy, heart block, and reentry.

Background Neutrophils are thought to be short‐lived first responders to tissue injuries such as myocardial infarction (MI), but little is known about their diversification or dynamics. Methods and Results We permanently ligated the left anterior descending coronary arteries of mice and performed single‐cell RNA sequencing and analysis of >28 000 neutrophil transcriptomes isolated from the heart, peripheral blood, and bone marrow of mice on days 1 to 4 after MI or at steady‐state. Unsupervised clustering of cardiac neutrophils revealed 5 major subsets, 3 of which originated in the bone marrow, including a late‐emerging granulocyte expressing SiglecF, a marker classically used to define eosinophils. SiglecF HI neutrophils represented ≈25% of neutrophils on day 1 and grew to account for >50% of neutrophils by day 4 post‐MI. Validation studies using quantitative polymerase chain reaction of fluorescent‐activated cell sorter sorted Ly6G + SiglecF HI and Ly6G + SiglecF LO neutrophils confirmed the distinct nature of these populations. To confirm that the cells were neutrophils rather than eosinophils, we infarcted GATA‐deficient mice (∆dblGATA) and observed similar quantities of infiltrating Ly6G + SiglecF HI cells despite marked reductions of conventional eosinophils. In contrast to other neutrophil subsets, Ly6G + SiglecF HI neutrophils expressed high levels of Myc‐regulated genes, which are associated with longevity and are consistent with the persistence of this population on day 4 after MI. Conclusions Overall, our data provide a spatial and temporal atlas of neutrophil specialization in response to MI and reveal a dynamic proinflammatory cardiac Ly6G + SigF + (Myc + NFϰB + ) neutrophil that has been overlooked because of negative selection.

In the adult heart, the epicardium becomes activated after injury, contributing to cardiac healing by secretion of paracrine factors. Here we analyzed by single-cell RNA sequencing combined with RNA in situ hybridization and lineage tracing of WT1+ cells the cellular composition, location, and hierarchy of epicardial stromal cells (EpiSC) in comparison to activated myocardial fibroblasts/stromal cells in infarcted mouse hearts. We identified 11 transcriptionally distinct EpiSC populations, that can be classified in three groups each containing a cluster of proliferating cells. Two groups expressed cardiac specification makers and sarcomeric proteins suggestive of cardiomyogenic potential. Transcripts of HIF-1α and HIF-responsive genes were enriched in EpiSC consistent with an epicardial hypoxic niche. Expression of paracrine factors was not limited to WT1+ cells but was a general feature of activated cardiac stromal cells. Our findings provide the cellular framework by which myocardial ischemia may trigger in EpiSC the formation of cardioprotective/regenerative responses.

Ischaemic heart disease is the world’s leading cause of mortality. Survival rates from acute myocardial infarction (MI) have improved in recent years; however, this has led to an increase in the prevalence of heart failure (HF) due to chronic remodelling of the infarcted myocardium, for which treatment options remain poor. We have previously shown that inhibition of isoform 4 of the plasma membrane calcium ATPase (PMCA4) prevents chronic remodelling and HF development during pressure overload, through fibroblast mediated Wnt signalling modulation. Given that Wnt signalling also plays a prominent role during remodelling of the infarcted heart, this study investigated the effect of genetic and functional loss of PMCA4 on cardiac outcomes following MI. Neither genetic deletion nor pharmacological inhibition of PMCA4 affected chronic remodelling of the post-MI myocardium. This was the case when PMCA4 was deleted globally, or specifically from cardiomyocytes or fibroblasts. PMCA4-ablated hearts were however less prone to acute arrhythmic events, which may offer a slight survival benefit. Overall, this study demonstrates that PMCA4 inhibition does not affect chronic outcomes following MI.

Nanoscale therapeutics have promise for the administration of therapeutic small molecules and biologics to the heart following myocardial infarction. Directed delivery to the infarcted region of the heart using minimally invasive routes is critical to this promise. In this review, we will discuss the advances and design considerations for two nanoscale therapeutics engineered to target the infarcted heart, nanoparticles and adeno-associated viruses.

This study aimed at developing a formulation to link microscopic structure and macroscopic mechanics of a fibrous scaffold filled with a hydrogel for use as a tissue-engineered patch for local epicardial support of the infarcted heart. Mori-Tanaka mean field homogenisation, closed-cell foam mechanics and finite element (FE) methods were used to represent the macroscopic elastic modulus of the filled fibrous scaffold. The homogenised constitutive description of the scaffold was implemented for an epicardial patch in a FE model of a human cardiac left ventricle (LV) to assess effects of patching on myocardial mechanics and ventricular function in presences of an infarct. The macroscopic elastic modulus of the scaffold was predicted to be 0.287 MPa with the FE method and 0.290 MPa with the closed-cell model for the realistic fibre structure of the scaffold, and 0.108 and 0.540 MPa with mean field homogenization for randomly oriented and completely aligned fibres. Epicardial patching was predicted to reduce maximum myocardial stress in the infarcted LV from 19 kPa (no patch) to 9.5 kPa (patch), and to increase the ventricular ejection fraction from 40% (no patch) to 43% (patch). The predictions of the macroscopic elastic modulus of the realistic scaffold with the FE and the closed-cell model agreed well, and were bound by the mean field homogenisation prediction for random and fully aligned fibre orientation of the scaffold. This study demonstrates the feasibility of homogenization techniques to represent complex multiscale structural features in an simplified but meaningful manner.

Repair of the infarcted heart is dependent on inflammation-driven activation of myofibroblasts (MFs) and subsequent formation of a scar. Though pericytes have been implicated in injury-associated fibroblast activation in several organs, their potential role in cardiac repair and fibrosis has not been studied. We hypothesized that myocardial infarction (MI) may induce pericyte activation, contributing to repair through pericyte to MF conversion, secretion of fibrogenic mediators, or regulation of angiogenesis. In order to test the hypothesis, we generated pericyte/fibroblast reporter mice (NG2 DsRed ;PDGFRα GFP ). In normal myocardium, NG2 labeled peri-endothelial mural cells that coexpressed PDGFRβ, whereas PDGFRα identified interstitial cells with fibroblast characteristics. Pericytes and fibroblasts had distinct transcriptomic profiles: NG2+/PDGFRα- pericytes expressed αSMA and low amounts of extracellular matrix (ECM) genes, whereas PDGFRα+/NG2- fibroblasts synthesized collagens. Pericyte rarefaction was noted in the necrotic core 3 days after non-reperfused MI. 3-7 days post MI, expansion of the NG2+ population in the infarct zone was associated with emergence of non-mural NG2+/αSMA+ cells with MF characteristics. FACS-sorted NG2+/PDGFRα- cells from 7-day infarcts expressed higher levels of ColIα2 (7.2±1.0-fold) and ColIIIα1 (8.9±1.14-fold), when compared to NG2+/PDGFRα- cells from normal hearts. NG2+ cells had high mRNA levels of integrins α1, αV, β1, and β5, and of MMP14, reflecting an activated migratory phenotype. To examine whether expression of ECM genes by infarct pericytes is due to fibroblast conversion, we did lineage tracing studies using NG2CreER TM ;Rosa tdTomato mice bred with the PDGFRα GFP line for reliable fibroblast identification. 7 days post MI, 5.7%±1.04 of PDGFRα+ fibroblasts were derived from NG2+ cells. Also, αSMA staining showed that 10.49%±2.73 of infarct MFs were derived from NG2+ lineage. The majority of mural cells wrapping neovessels were derived from NG2+ cells, suggesting a role for resident pericytes in infarct angiogenesis. In conclusion, upon MI, pericytes become activated and contribute to repair by undergoing conversion to a subset of myofibroblasts and by coating infarct neovessels.

Funding Acknowledgements National Institute for Health Research; British Heart Foundation; and The Wellcome Trust and Engineering and Physical Sciences Research Council. Background Delayed afterdepolarizations (DADs) due to spontaneous calcium (Ca) release (SCR) events from the sarcoplasmic reticulum have been implicated with a variety of arrhythmias. Such SCR events have also been reported in cells that survive in the infarct border zone (BZ). While the potential of Ca-mediated DADs to become suprathreshold and propagate in the form of ectopic beats has been well characterized, the role of subthreshold DADs in arrhythmia formation in the infarcted heart remains to be elucidated. Purpose To use computational modelling to investigate whether subthreshold Ca-mediated DADs may form a substrate for conduction block and reentry in the BZ. Our hypothesis is that subthreshold DADs can hinder local tissue excitability in critical infarct BZ regions by inactivating the fast sodium current (INa), leading to temporary unidirectional conduction block providing a trigger for arrhythmogenesis. Methods We developed an idealized infarct model of the left ventricle. The infarct region consisted of a non-conducting scar transcended by an isthmus of cells that survived myocardial infarction (border zone). These cells were made prone to Ca-mediated DADs described by a phenomenological model of SCR events. The model was pre-paced at the apex followed by a 1500ms-pacing pause to see whether DADs would emerge. An extra beat with a longer coupling interval (CI) was then applied. The following electrophysiological changes resulting from remodeling processes in the isthmus were simulated to assess their contribution to the arrhythmogenic potential of subthreshold DADs: INa loss-of-function due to a (2.5mV and 5mV) negative-shift in the steady-state channel inactivation; 50% reduction in tissue conductivity; and increased levels of fibrosis (up to 50%). Results On average, Ca-mediated DADs reached their maximum value 1065ms after the last paced beat (Fig. A). Despite this, in the default electrophysiological setup, simulations with extra beats with 1000ms > CI > 1100ms did not result in conduction block in any of the experiments. When repeated with combined changes of reduced tissue conductivity and fibrosis, subthreshold DADs were still unable to create a substrate for block. However, when combined with a 5mV-shift in INa inactivation, block at isthmus’ mouth proximal to the stimulus site was detected for extra beats 1010 ms ≥ CI ≥ 1070ms (see Fig. B). The cause of block was due to a subthreshold DAD occurring just prior to the arrival of the extra beat. All blocked beats degenerated into reentry. Conclusions Under most physiological conditions, subthreshold DADs are unlikely to provide a substrate for unidirectional block. However, under conditions of decreased excitability, subthreshold DADs can hinder tissue excitability in the infarcted region leading to conduction block and reentry. Abstract Figure. DAD-mediated conduction block in the BZ

The lymphatic system plays crucial roles in regulating fluid homeostasis, immune surveillance, and lipid transport. As is in most of the body's organs, the heart possesses an extensive lymphatic network. Moreover, a robust lymphangiogenic response has been shown to take place following myocardial infarction, highlighting cardiac lymphatics as potential targets for therapeutic intervention. Yet, the unique molecular properties and functions of the heart's lymphatic system have only recently begun to be addressed. In this review, we discuss the mechanisms underlying the formation and growth of cardiac lymphatics during embryonic development and describe their characteristics across species. We further summarize recent findings highlighting diverse cellular origins for cardiac lymphatic endothelial cells and how they integrate to form a single functional lymphatic network. Finally, we outline novel therapeutic avenues aimed at enhancing lymphatic vessel formation and integrity following cardiac injury, which hold great promise for promoting healing of the infarcted heart.

Gold nanostructures can be incorporated into macroporous scaffolds to increase the matrix conductivity and enhance the electrical signal transfer between cardiac cells. Here we report a simple approach for fabricating 3-dimensional (3D) gold nanoparticle (NP)-based fibrous scaffolds, for engineering functional cardiac tissues generating a strong contraction force. A polycaprolactone–gelatin mixture was electrospun to obtain fibrous scaffolds with an average fiber diameter of 250 nm. In a facile method, gold NPs were evaporated on the surface of the fibers, creating nanocomposites with a nominal gold thickness of 2, 4, and 14 nm. Compared to pristine scaffolds, cardiac cells seeded on the nano-gold scaffolds assembled into more elongated and aligned tissues. The gold NPs on the fibers were able to maintain the ratio of cardiomyocytes to fibroblasts in the culture, to encourage the growth of cardiomyocytes with significantly higher aspect ratio, and promote massive cardiac sarcomeric actinin expression. Finally, engineering cardiac tissues within gold NP-based scaffolds exhibited significantly higher contraction amplitudes and rates, as compared to scaffolds without gold. We envision that cardiac tissues engineered within these gold NP scaffolds can be used to improve the function of the infarcted heart.

The use of injectable scaffolds to repair the infarcted heart is receiving great interest. Thermosensitive polymers, in situ polymerization, in situ cross-linking, and self-assembling peptides are the most investigated approaches to obtain injectability.Aim of the present work was the preparation and characterization of a novel bioactive scaffold, in form of injectable microspheres, for cardiac repair. Gellan/gelatin microspheres were prepared by a water-in-oil emulsion and loaded by adsorption with Insulin-like growth factor 1 to promote tissue regeneration. Obtained microspheres underwent morphological, physicochemical and biological characterization, including cell culture tests in static and dynamic conditions and in vivo tests. Morphological analysis of the microspheres showed a spherical shape, a microporous surface and an average diameter of 66 ± 17µm (under dry conditions) and 123 ± 24 µm (under wet conditions). Chemical Imaging analysis pointed out a homogeneous distribution of gellan, gelatin and Insulin-like growth factor-1 within the microsphere matrix. In vitro cell culture tests showed that the microspheres promoted rat cardiac progenitor cells adhesion, and cluster formation. After dynamic suspension culture within an impeller-free bioreactor, cells still adhered to microspheres, spreading their cytoplasm over microsphere surface. Intramyocardial administration of microspheres in a cryoinjury rat model attenuated chamber dilatation, myocardial damage and fibrosis and improved cell homing.Overall, the findings of this study confirm that the produced microspheres display morphological, physicochemical, functional and biological properties potentially adequate for future applications as injectable scaffold for cardiac tissue engineering.

Although cardiac patches hold a promise for repairing the infarcted heart, their integration with the myocardium by sutures may cause further damage to the diseased organ. To address this issue, we developed facile and safe, suture-free technology for the attachment of engineered tissues to organs. Here, nanocomposite scaffolds comprised of albumin electrospun fibers and gold nanorods (AuNRs) were developed. Cardiac cells were seeded within the scaffolds and assembled into a functioning patch. The engineered tissue was then positioned on the myocardium and irradiated with a near IR laser (808 nm). The AuNRs were able to absorb the light and convert it to thermal energy, which locally changed the molecular structure of the fibrous scaffold, and strongly, but safely, attached it to the wall of the heart. Such hybrid biomaterials can be used in the future to integrate any engineered tissue with any defected organs, while minimizing the risk of additional injury for the patient, caused by the conventional stitching methods.

Transplantations of various stem cells or their progeny have repeatedly improved cardiac performance in animal models of myocardial injury; however, the benefits observed in clinical trials have been generally less consistent. Some of the recognized challenges are poor engraftment of implanted cells and, in the case of human cardiomyocytes, functional immaturity and lack of electrical integration, leading to limited contribution to the heart’s contractile activity and increased arrhythmogenic risks. Advances in tissue and genetic engineering techniques are expected to improve the survival and integration of transplanted cells, and to support structural, functional, and bioenergetic recovery of the recipient hearts. Specifically, application of a prefabricated cardiac tissue patch to prevent dilation and to improve pumping efficiency of the infarcted heart offers a promising strategy for making stem cell therapy a clinical reality.

Nanoliposomes are type of nano-sized vesicles made of bi-layered phospholipid membranes with an aqueous interior. They have been demonstrated to deliver several materials like low molecular weight drugs, imaging agents, peptides, proteins, and nucleic acids. Nanoliposomes have been demonstrated to slowly release an encapsulated drug, thereby leading to sustained exposure to target region and improved efficacy. This ability of nano-liposomes can be harnessed to deliver therapeutic agents precisely to the infarcted heart. Accordingly, this article will review recent developments in the application of nano liposomes and nanoparticles as drug delivery systems to treat cardiovascular related disorders such as atherosclerosis, restenosis and myocardial infarction.

The adult myocardium has a limited regenerative capacity following heart injury and the lost cells are primarily replaced by fibrotic scar tissue. Suboptimal efficiency of current clinical therapies to resurrect the infarcted heart results in injured heart enlargement and remodeling to maintain its physiological functions. These remodeling processes ultimately leads to ischemic cardiomyopathy and heart failure (HF). Recent therapeutic approaches (e.g., regenerative and nanomedicine) have shown promise to prevent HF post-myocardial infarction in animal models. However, these preclinical, clinical, and technological advancements have yet to yield substantial enhancements in the survival rate and quality of life of patients with severe ischemic injuries. This could be attributed largely to the considerable gap in knowledge between clinicians and nano-bioengineers. Development of highly effective cardiac regenerative therapies requires connecting and coordinating multiple fields, including cardiology, cellular and molecular biology, biochemistry and chemistry, and mechanical and materials sciences, among others. This review is particularly intended to bridge the knowledge gap between cardiologists and regenerative nanomedicine experts. Establishing this multidisciplinary knowledge base may help pave the way for developing novel, safer, and more effective approaches that will enable the medical community to reduce morbidity and mortality in HF patients.

Introduction: Ischemic Heart Disease (IHD), the leading cause of death in the world, is often initiated by myocardial infarction (MI). In mice, death typically occurs on day 4 after acute MI is induced by coronary artery ligation. At this time the interferon regulatory factor 3 (IRF3)/type I interferon (IFN) pathway is activated almost exclusively in mononuclear cells in the infarct. We have shown that the genetic or pharmacologic disruption of critical components of this innate immune pathway reduces inflammation, limits ventricular dilation and contractile dysfunction, and improves survival. Hypothesis: Treatment with the IFNAR neutralizing antibody will bias the differentiation of cardiac macrophages and in doing so establish a cardioprotective environment in the infarct. Methods: We conducted single cell RNA-sequencing analysis of mononuclear cells isolated from infarct tissue collected on day 4 after MI from mice that had been treated with an IFNAR neutralizing antibody (Ab) as well as untreated mice. Results: Analyses demonstrate the presence of 3 core cell populations in the untreated sample: monocytes expressing Plac8 and Ms4a4c , phagocytic macrophages expressing Mrc1 and Ccl8 , and reparative macrophages expressing Arg1 and Fn1 . In addition, the dramatic co-expression of related genes in response to environmental stimuli, such as secreted type I IFNs or reactive oxygen species (ROS), resulted in the formation of two more clusters of cells. One cluster was marked by the expression of interferon stimulated genes. The other was marked by the expression of an established anti-oxidant transcriptional program. Anti-interferon therapy with IFNAR Ab inhibited both the interferon stimulated genes and the anti-oxidative transcriptional program. Conclusion: We have defined the differentiation patterns of monocytes and macrophages within the infarcted heart on day 4 after MI. Treatment with the IFNAR Ab after MI alters the composition of macrophage subsets.

Type 2 immunity participates in the pathogeneses of helminth infection and allergic diseases. Emerging evidence indicates that the components of type 2 immunity are also involved in maintaining metabolic hemostasis and facilitating the healing process after tissue injury. Numerous preclinical studies have suggested regulation of type 2 immunity-related cytokines, such as interleukin-4, -13, and -33, and cell types, such as M2 macrophages, mast cells, and eosinophils, affects cardiac functions after myocardial infarction (MI), providing new insights into the importance of immune modulation in the infarcted heart. This review provides an overview of the functions of these cytokines and cells in the setting of MI as well as their potential to predict the severity and prognosis of MI.

Altres ajuts:The present study was supported by the EU Joint Programming Initiative 'A Healthy Diet for a Healthy Life' (JPI HDHL INTIMIC-085), Generalitat Valenciana (GV/2018/116), INCLIVA and Universitat de Valencia (program VLC-BIOCLINIC 20-nanomIRM-2016A). Delayed enhancement cardiovascular magnetic resonance (MR) is the gold-standard for non-invasive assessment after myocardial infarction (MI). MR microscopy (MRM) provides a level of detail comparable to the macro objective of light microscopy. We used MRM and correlative histopathology to identify infarct and remote tissue in contrast agent-free multi-sequence MRM in swine MI hearts. One control group (n = 3 swine) and two experimental MI groups were formed: 90 min of ischemia followed by 1 week (acute MI = 6 swine) or 1 month (chronic MI = 5 swine) reperfusion. Representative samples of each heart were analysed by contrast agent-free multi-sequence (T1-weighting, T2-weighting, T2*-weighting, T2-mapping, and T2*-mapping). MRM was performed in a 14-Tesla vertical axis imager (Bruker-AVANCE 600 system). Images from MRM and the corresponding histopathological stained samples revealed differences in signal intensities between infarct and remote areas in both MI groups (p-value < 0.001). The multivariable models allowed us to precisely classify regions of interest (acute MI: specificity 92% and sensitivity 80%; chronic MI: specificity 100% and sensitivity 98%). Probabilistic maps based on MRM images clearly delineated the infarcted regions. As a proof of concept, these results illustrate the potential of MRM with correlative histopathology as a platform for exploring novel contrast agent-free MR biomarkers after MI.

Background The mechanisms underpinning the regenerative capabilities of mesenchymal stem cells (MSC) were originally thought to reside in their ability to recognise damaged tissue and to differentiate into specific cell types that would replace defective cells. However, recent work has shown that molecules produced by MSCs (secretome), particularly those packaged in extracellular vesicles (EVs), rather than the cells themselves are responsible for tissue repair. Methods Here we have produced a secretome from adipose-derived mesenchymal stem cells (ADSC) that is free of exogenous molecules by incubation within a saline solution. Various in vitro models were used to evaluate the effects of the secretome on cellular processes that promote tissue regeneration. A cardiotoxin-induced skeletal muscle injury model was used to test the regenerative effects of the whole secretome or isolated extracellular vesicle fraction in vivo. This was followed by bioinformatic analysis of the components of the protein and miRNA content of the secretome and finally compared to a secretome generated from a secondary stem cell source. Results Here we have demonstrated that the secretome from adipose-derived mesenchymal stem cells shows robust effects on cellular processes that promote tissue regeneration. Furthermore, we show that the whole ADSC secretome is capable of enhancing the rate of skeletal muscle regeneration following acute damage. We assessed the efficacy of the total secretome compared with the extracellular vesicle fraction on a number of assays that inform on tissue regeneration and demonstrate that both fractions affect different aspects of the process in vitro and in vivo. Our in vitro, in vivo, and bioinformatic results show that factors that promote regeneration are distributed both within extracellular vesicles and the soluble fraction of the secretome. Conclusions Taken together, our study implies that extracellular vesicles and soluble molecules within ADSC secretome act in a synergistic manner to promote muscle generation. Electronic supplementary material The online version of this article (10.1186/s13287-019-1213-1) contains supplementary material, which is available to authorized users.

c-Kitpos cardiac progenitor cells (CPCs) represent a successful approach in healing the infarcted heart and rescuing its mechanical function, but electrophysiological consequences are uncertain. CPC mobilization promoted by hepatocyte growth factor (HGF) and IGF-1 improved electrogenesis in myocardial infarction (MI). We hypothesized that locally delivered CPCs supplemented with HGF + IGF-1 (GFs) can concur in ameliorating electrical stability of the regenerated heart. Adult male Wistar rats (139 rats) with 4-wk-old MI or sham conditions were randomized to receive intramyocardial injection of GFs, CPCs, CPCs + GFs, or vehicle (V). Enhanced green fluorescent protein-tagged CPCs were used for cell tracking. Vulnerability to stress-induced arrhythmia was assessed by telemetry-ECG. Basic cardiac electrophysiological properties were examined by epicardial multiple-lead recording. Hemodynamic function was measured invasively. Hearts were subjected to anatomical, morphometric, immunohistochemical, and molecular biology analyses. Compared with V and at variance with individual CPCs, CPCs + GFs approximately halved arrhythmias in all animals, restoring cardiac anisotropy toward sham values. GFs alone reduced arrhythmias by less than CPCs + GFs, prolonging ventricular refractoriness without affecting conduction velocity. Concomitantly, CPCs + GFs reactivated the expression levels of Connexin-43 and Connexin-40 as well as channel proteins of key depolarizing and repolarizing ion currents differently than sole GFs. Mechanical function and anatomical remodeling were equally improved by all regenerative treatments, thus exhibiting a divergent behavior relative to electrical aspects. Conclusively, we provided evidence of distinctive antiarrhythmic action of locally injected GF-supplemented CPCs, likely attributable to retrieval of Connexin-43, Connexin-40, and Cav1.2 expression, favoring intercellular coupling and spread of excitation in mended heart.

Injectable biomaterials are promising as new therapies to treat myocardial infarction (MI). One useful property of biomaterials is the ability to protect and sustain release of therapeutic payloads. In order to create a platform for optimizing the release rate of cardioprotective molecules we utilized the tunable degradation of acetalated dextran (AcDex). We created microparticles with three distinct degradation profiles and showed that the consequent protein release profiles could be modulated within the infarcted heart. This enabled us to determine how delivery rate impacted the efficacy of a model therapeutic, an engineered hepatocyte growth factor fragment (HGF-f). Our results showed that the cardioprotective efficacy of HGF-f was optimal when delivered over three days post-intramyocardial injection, yielding the largest arterioles, fewest apoptotic cardiomyocytes bordering the infarct and the smallest infarcts compared to empty particle treatment four weeks after injection. This work demonstrates the potential of using AcDex particles as a delivery platform to optimize the time frame for delivering therapeutic proteins to the heart.

Allogeneic mesenchymal stem cells (MSCs) derived from bone marrow are considered to ideal candidate cell type for myocardial repair after a cardiac injury. However, recent reviews of allogeneic MSCs based clinical trials reported that transplanted cells were not able to survive for long term in the heart. Therefore, strategies to improve survival of transplanted cells would preserve the benefits of allogeneic MSCs therapy for heart repair. Recently, graphene oxide (GO) based biomaterials are being widely investigated for their ability to improve survival of transplanted MSCs in the heart. However, the presence of reactive oxygen functional groups (-OH, -COOH) in the structure of GO increases oxidative stress and activate cell death pathways, which impairs the benefits of GO based biomaterials. Therefore, to improve benefits of GO for stem cell delivery, we synthesized a thermosensitive hydrogel by conjugating GO with chitosan (CS). Our fourier transformed infrared spectrum (FTIR) analysis revealed an interaction between oxygen functional groups of GO and amino (-NH2) groups of chitosan. Our data demonstrate that CS masked the reactive functional groups of GO and prevented GO mediated ROS induction. This novel chitosan-graphene oxide (CS-GO) composite exhibited optimal porosity for cell conjugation and retention. Furthermore, CS-GO biomaterial was cyto-compatible as there was no cytotoxicity caused by CS-GO to MSCs. Furthermore, CS-GO also prevented hypoxia induced apoptosis of MSCs. We employed this novel biomaterial to deliver allogeneic MSCs to the infarcted heart in a rat model of myocardial infarction. After 5 weeks of cell transplantation, there was a significant improvement in the survival of implanted MSCs and cardiac function in CS-GO group. Therefore, conjugation of GO with chitosan prevented GO mediated ROS generation and cell death. Also, CS-GO mediated delivery of allogeneic MSCs to infarcted heart improved the survival of transplanted cells and preserved cardiac function.