Adult Cardiac Cells Research Articles

Mechanical load regulates the proliferation of multiple cell types in the heart

Abstract Funding Acknowledgements None. Introduction The adult mammalian heart has a poor regenerative and angiogenic potential. At the same time, both primary cardiac tumors and cardiac metastases are extremely rare. This suggests that shared mechanisms may halt the proliferation of cardiac (both cardiomyocyte and endothelial) and cancer cells. Among the mechanisms that have been claimed to be responsible for the loss of proliferative potential of cardiomyocytes early after birth is a sudden increase in mechanical load. Purpose Here, we investigate whether alterations in mechanical load affect the proliferation of multiple cell types in the heart. Methods Adult heart unloading in vivo was achieved using a mouse model of heterotopic heart transplantation, followed by EdU injection to label proliferating cells. Engineered heart tissues (EHTs) were generated using neonatal cardiomyocytes and fibroblasts, with a modified protocol allowing to finely tune mechanical load. Primary cardiac endothelial cells and multiple types of cancer cells (lung, melanoma and colon cancer) along with adult cardiac endothelial cells were included in EHTs. Results In line with our hypothesis, unloaded, heterotransplanted hearts showed a significant number of proliferating cardiomyocytes and endothelial cells, which were almost absent in native hearts. While cancer cells did not grow in native hearts, they massively proliferated in unloaded hearts. Consistent with in vivo results, mechanical unloading in EHTs significantly increased the percentage of EdU-positive cardiomyocytes, endothelial and cancer cells. In contrast, increasing afterload reduced cell proliferation in all cell types. Mechanistically, we identified Nesprin2 as an essential factor in sensing mechanical load and translating it into a cell proliferation arrest. Conclusions Overall, these data indicate that variations in mechanical load have a dramatic effect on the proliferation of multiple cell types within the heart tissue, including cardiomyocytes, endothelial and cancer cells and that Nesprin2 is required to mediate this effect.Unloading in the heartUnloading in EHT

Tailup expression in Drosophila larval and adult cardiac valve cells.

In Drosophila larvae, the direction of blood flow within the heart tube, as well as the diastolic filling of the posterior heart chamber, is regulated by a single cardiac valve. This valve is sufficient to close the heart tube at the junction of the ventricle and the aorta and is formed by only two cells; both are integral parts of the heart tube. The valve cells regulate hemolymph flow by oscillating between a spherical and a flattened cell shape during heartbeats. At the spherical stage, the opposing valve cells close the heart lumen. The dynamic cell shape changes of valve cells are supported by a dense, criss-cross orientation of myofibrils and the presence of the valvosomal compartment, a large intracellular cavity. Both structures are essential for the valve cells' function. In a screen for factors specifically expressed in cardiac valve cells, we identified the transcription factor Tailup. Knockdown of tailup causes abnormal orientation and differentiation of cardiac muscle fibers in the larval aorta and inhibits the formation of the ventral longitudinal muscle layer located underneath the heart tube in the adult fly and affects myofibrillar orientation of valve cells. Furthermore, we have identified regulatory sequences of tup that control the expression of tailup in the larval and adult valve cells.

Abstract P2025: BET Bromodomain Proteins Are Necessary For TGFβ2-Mediated Myofibroblast Activation

Fibrosis is a key feature of dilated cardiomyopathy (DCM). Stress signaling triggers the activation of quiescent fibroblasts to myofibroblasts. Bromodomain and extraterminal (BET) epigenetic reader proteins integrate stress-responsive signaling. We explored the role of BETs on distinct inflammatory signaling pathways in cardiac fibroblasts. Fibroblasts were isolated from wild type hearts using the Langendorff procedure. Cell cultures were treated with vehicle, the pan-BET inhibitor JQ1, or siRNA targeting Smad3 or Rela (to inhibit TGFβ and NFκB pathways, respectively) followed by TGFβ2 stimulation. Gene expression was assessed by QPCR, α-smooth muscle actin (αSMA) was quantified by western blot, and NFκB transcription was evaluated using a dual luciferase reporter assay. The interaction between BRD4 and SMAD3 was assessed by coimmunoprecipitation (coIP) in immortalized adult mouse cardiac fibroblast cell cultures treated with TGFβ2, JQ1, or vehicle. TGFβ2-induced fibroblast activation was ablated by JQ1 and Smad3 siRNA (reduced expression of Acta2, Col3a1, Postn and αSMA). CoIP revealed a direct interaction between BRD4 and SMAD3 in fibroblasts, which increased 2.2-fold (p<0.05) after TGFβ2 stimulation. By contrast, Rela siRNA alone induced myofibroblast gene expression ( Acta2 , 6.9-fold; Col3a1 , 7.0-fold; Postn , 11.2-fold; p<0.05 vs. vehicle for all), and increased αSMA levels (2.2-fold; p<0.05). This effect of Rela siRNA was similar to that observed with TGFβ2 stimulation. Further, TGFβ2 inhibited NFκB-driven gene transcription (-2.9-fold; p<0.05 vs. vehicle). These data demonstrate that BRD4 is a critical co-activator of TGFβ2-SMAD3 signaling and cardiac fibroblast activation. We also found that NFκB signaling is downregulated by TGFβ2, suggesting that NFκB may modulate inflammatory gene network activation.

Comprehensive Expression Analysis of Cardiac Fibroblast Growth Factor 23 in Health and Pressure-induced Cardiac Hypertrophy.

Enhanced fibroblast growth factor 23 (FGF23) is associated with left ventricular hypertrophy (LVH) in patients with chronic kidney and heart disease. Experimentally, FGF23 directly induces cardiac hypertrophy and vice versa cardiac hypertrophy stimulates FGF23. Besides the bone, FGF23 is expressed by cardiac myocytes, whereas its synthesis in other cardiac cell types and its paracrine role in the heart in health and disease is unknown. By co-immunofluorescence staining of heart tissue of wild-type mice, we show that Fgf23 is expressed by cardiac myocytes, fibroblasts and endothelial cells. Cardiac Fgf23 mRNA and protein level increases from neonatal to six months of age, whereas no age-related changes in bone Fgf23 mRNA expression were noted. Cardiac myocyte-specific disruption of Fgf23 using Cre-LoxP system (Fgf23fl/fl/cre+) caused enhanced mortality, but no differences in cardiac function or structure. Although pressure overload-induced cardiac hypertrophy induced by transverse aortic constriction (TAC) resulted in a slightly worse phenotype with a more severe reduced ejection fraction, higher end-systolic volume and more enlarged systolic LV diameter in Fgf23fl/fl/cre+ mice compared to controls, this was not translated to any worse cellular hypertrophy, fibrosis or chamber remodeling. TAC induced Fgf23 mRNA expression in whole cardiac tissue in both genotypes. Interestingly, co-immunofluorescence staining revealed enhanced Fgf23 synthesis in cardiac fibroblasts and endothelial cells but not in cardiac myocytes. RNA sequencing of isolated adult cardiac myocytes, cardiac fibroblasts and endothelial cells confirmed significantly higher Fgf23 transcription in cardiac fibroblasts and endothelial cells after TAC. Our data indicate that Fgf23 is physiologically expressed in various cardiac cell types and that cardiac fibroblasts and endothelial cells might be an important source of FGF23 in pathological conditions. In addition, investigations in Fgf23fl/fl/cre+ mice suggest that cardiac myocyte-derived FGF23 is needed to maintain cardiac function during pressure overload.

Tbx5 navigates through the labyrinth of adult cardiac regeneration

Abstract Background Heart failure is the major cause of death and morbidity in industrialized countries with an estimated 23 million people affected per year, representing 30% of all global deaths. Injury to the adult mammalian cardiac muscle, often leads to a heart attack due to irreversible loss of a large number of cardiomyocytes (CM) and other cardiac interstitial cells, creating an unmet need for identifying a cardiac progenitor cell (CPC) population for cardiac replenishment. In contrast, amphibians and neonatal rodents possess the ability to regenerate their heart upon injury. It has been suggested recently that idle cardiac regenerative mechanisms may be present in adult mammals, inhibited by exogenous cues, or lack of. Murine and human CPCs can be isolated through the expression of Pdgfra, Kdr, and our novel surface marker, Gfra2. In addition, the expression of the embryonic transcription factor TBX5, is paramount for differentiation towards a cardiomyocyte fate. Therefore, Tbx5-expressing CPCs could be an effective target for proof-of-concept studies in the heart repair field, inclined to pharmacological modulation in patients with ischemic heart disease. Purpose To characterise an adult Tbx5-expressing CPC population in the injured heart. Using a developmental approach to two adult heart injury murine models, we show that Tbx5-expressing CPC exist in the injured adult mammalian heart, with a molecular signature that strongly correlates with that of embryonic and neonatal CPCs. Methods A well-defined tamoxifen-induced Tbx5-Cre; Rosa26R-eYFP/eYFP transgenic mouse model was employed, where myocardial infarction (MI) was induced through reperfusion/ischemia or chemical injury. Cardiac cells expressing YFP+ cells were collected from the adult injured hearts five to seven days post-injury. Flow-cytometric and single-cell mRNA-seq analysis was performed in order to collect and compare those YFP+ cells to control adult uninjured cardiac interstitial cells, and early neonatal-derived CPCs. Results Immunohistochemical analysis indicated that YFP+ interstitial cells were mostly present in the injury site, but also in distal cardiac areas. Flow cytometric analysis of live cells pinpoint these YFP+ cells are part of a CPC-like population. Single-cell mRNA transcriptomic analysis revealed that YFP+ cells resemble early postnatal CPC. Yet, YFP+ cells never expressed CM markers in vivo, but they did differentiate into CM-like cells, in vitro. Conclusions Upon MI, the adult heart possess an interstitial cell population that transiently re-activates the pioneer cardiac embryonic transcription factor Tbx5. The Tbx5-expressing cell population transcriptomically resembles that of CPC, which could promote CM regeneration upon neonatal injury. We show that Tbx5 lies in the centre of a novel adult CPC population. The adult heart microenvironment may hinder mammalian CM regeneration through regulation of the Tbx5-induced cardiac gene program. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Hellenic Foundation for Research & Innovation

Kanglexin protects against cardiac fibrosis and dysfunction in mice by TGF-β1/ERK1/2 noncanonical pathway.

Cardiac fibrosis is a common pathological manifestation accompanied by various heart diseases, and antifibrotic therapy is an effective strategy to prevent diverse pathological processes of the cardiovascular system. We currently report the pharmacological evaluation of a novel anthraquinone compound (1,8-dihydroxy-6-methyl-9,10-anthraquinone-3-oxy ethyl succinate) named Kanglexin (KLX), as a potent cardioprotective agent with antifibrosis activity. Our results demonstrated that the administration of KLX by intragastric gavage alleviated cardiac dysfunction, hypertrophy, and fibrosis induced by transverse aortic constriction (TAC) surgical operation. Meanwhile, KLX administration relieved endothelial to mesenchymal transition of TAC mice. In TGF β1-treated primary cultured adult mouse cardiac fibroblasts (CFs) and human umbilical vein endothelial cells (HUVECs), KLX inhibited cell proliferation and collagen secretion. Also, KLX suppressed the transformation of fibroblasts to myofibroblasts in CFs. Further studies revealed that KLX-mediated cardiac protection was due to the inhibitory role of TGF-β1/ERK1/2 noncanonical pathway. In summary, our study indicates that KLX attenuated cardiac fibrosis and dysfunction of TAC mice, providing a potentially effective therapeutic strategy for heart pathological remodeling.

Strategies and Challenges to Improve Cellular Programming-Based Approaches for Heart Regeneration Therapy.

Limited adult cardiac cell proliferation after cardiovascular disease, such as heart failure, hampers regeneration, resulting in a major loss of cardiomyocytes (CMs) at the site of injury. Recent studies in cellular reprogramming approaches have provided the opportunity to improve upon previous techniques used to regenerate damaged heart. Using these approaches, new CMs can be regenerated from differentiation of iPSCs (similar to embryonic stem cells), the direct reprogramming of fibroblasts [induced cardiomyocytes (iCMs)], or induced cardiac progenitors. Although these CMs have been shown to functionally repair infarcted heart, advancements in technology are still in the early stages of development in research laboratories. In this review, reprogramming-based approaches for generating CMs are briefly introduced and reviewed, and the challenges (including low efficiency, functional maturity, and safety issues) that hinder further translation of these approaches into a clinical setting are discussed. The creative and combined optimal methods to address these challenges are also summarized, with optimism that further investigation into tissue engineering, cardiac development signaling, and epigenetic mechanisms will help to establish methods that improve cell-reprogramming approaches for heart regeneration.

MiR-96 and miR-183 differentially regulate neonatal and adult postinfarct neovascularization.

Following myocardial infarction (MI), the adult heart has minimal regenerative potential. Conversely, the neonatal heart can undergo extensive regeneration, and neovascularization capacity was hypothesized to contribute to this difference. Here, we demonstrate the higher angiogenic potential of neonatal compared with adult mouse cardiac endothelial cells (MCECs) in vitro and use this difference to identify candidate microRNAs (miRs) regulating cardiac angiogenesis after MI. miR expression profiling revealed miR-96 and miR-183 upregulation in adult compared with neonatal MCECs. Their overexpression decreased the angiogenic potential of neonatal MCECs in vitro and prevented scar resolution and neovascularization in neonatal mice after MI. Inversely, their inhibition improved the angiogenic potential of adult MCECs, and miR-96/miR-183–KO mice had increased peri-infarct neovascularization. In silico analyses identified anillin (ANLN) as a direct target of miR-96 and miR-183. In agreement, Anln expression declined following their overexpression and increased after their inhibition in vitro. Moreover, ANLN expression inversely correlated with miR-96 expression and age in cardiac ECs of cardiovascular patients. In vivo, ANLN+ vessels were enriched in the peri-infarct area of miR-96/miR-183–KO mice. These findings identify miR-96 and miR-183 as regulators of neovascularization following MI and miR-regulated genes, such as anillin, as potential therapeutic targets for cardiovascular disease.

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart.

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

Amorphous SiO2 nanoparticles promote cardiac dysfunction via the opening of the mitochondrial permeability transition pore in rat heart and human cardiomyocytes

BackgroundSilica nanoparticles (nanoSiO2) are promising systems that can deliver biologically active compounds to tissues such as the heart in a controllable manner. However, cardiac toxicity induced by nanoSiO2 has been recently related to abnormal calcium handling and energetic failure in cardiomyocytes. Moreover, the precise mechanisms underlying this energetic debacle remain unclear. In order to elucidate these mechanisms, this article explores the ex vivo heart function and mitochondria after exposure to nanoSiO2.ResultsThe cumulative administration of nanoSiO2 reduced the mechanical performance index of the rat heart with a half-maximal inhibitory concentration (IC50) of 93 μg/mL, affecting the relaxation rate. In isolated mitochondria nanoSiO2 was found to be internalized, inhibiting oxidative phosphorylation and significantly reducing the mitochondrial membrane potential (ΔΨm). The mitochondrial permeability transition pore (mPTP) was also induced with an increasing dose of nanoSiO2 and partially recovered with, a potent blocker of the mPTP, Cyclosporine A (CsA). The activity of aconitase and thiol oxidation, in the adenine nucleotide translocase, were found to be reduced due to nanoSiO2 exposure, suggesting that nanoSiO2 induces the mPTP via thiol modification and ROS generation. In cardiac cells exposed to nanoSiO2, enhanced viability and reduction of H2O2 were observed after application of a specific mitochondrial antioxidant, MitoTEMPO. Concomitantly, CsA treatment in adult rat cardiac cells reduced the nanoSiO2-triggered cell death and recovered ATP production (from 32.4 to 65.4%). Additionally, we performed evaluation of the mitochondrial effect of nanoSiO2 in human cardiomyocytes. We observed a 40% inhibition of maximal oxygen consumption rate in mitochondria at 500 μg/mL. Under this condition we identified a remarkable diminution in the spare respiratory capacity. This data indicates that a reduction in the amount of extra ATP that can be produced by mitochondria during a sudden increase in energy demand. In human cardiomyocytes, increased LDH release and necrosis were found at increased doses of nanoSiO2, reaching 85 and 48%, respectively. Such deleterious effects were partially prevented by the application of CsA. Therefore, exposure to nanoSiO2 affects cardiac function via mitochondrial dysfunction through the opening of the mPTP.ConclusionThe aforementioned effects can be partially avoided reducing ROS or retarding the opening of the mPTP. These novel strategies which resulted in cardioprotection could be considered as potential therapies to decrease the side effects of nanoSiO2 exposure.

Human Induced Pluripotent Stem Cells Derived from a Cardiac Somatic Source: Insights for an In-Vitro Cardiomyocyte Platform.

Reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs) has revolutionized the complex scientific field of disease modelling and personalized therapy. Cardiac differentiation of human iPSCs into cardiomyocytes (hiPSC-CMs) has been used in a wide range of healthy and disease models by deriving CMs from different somatic cells. Unfortunately, hiPSC-CMs have to be improved because existing protocols are not completely able to obtain mature CMs recapitulating physiological properties of human adult cardiac cells. Therefore, improvements and advances able to standardize differentiation conditions are needed. Lately, evidences of an epigenetic memory retained by the somatic cells used for deriving hiPSC-CMs has led to evaluation of different somatic sources in order to obtain more mature hiPSC-derived CMs.

Adaptation within embryonic and neonatal heart environment reveals alternative fates for adult c-kit+ cardiac interstitial cells.

Cardiac interstitial cells (CICs) perform essential roles in myocardial biology through preservation of homeostasis as well as response to injury or stress. Studies of murine CIC biology reveal remarkable plasticity in terms of transcriptional reprogramming and ploidy state with important implications for function. Despite over a decade of characterization and in vivo utilization of adult c‐Kit+ CIC (cCIC), adaptability and functional responses upon delivery to adult mammalian hearts remain poorly understood. Limitations of characterizing cCIC biology following in vitro expansion and adoptive transfer into the adult heart were circumvented by delivery of the donated cells into early cardiogenic environments of embryonic, fetal, and early postnatal developing hearts. These three developmental stages were permissive for retention and persistence, enabling phenotypic evaluation of in vitro expanded cCICs after delivery as well as tissue response following introduction to the host environment. Embryonic blastocyst environment prompted cCIC integration into trophectoderm as well as persistence in amniochorionic membrane. Delivery to fetal myocardium yielded cCIC perivascular localization with fibroblast‐like phenotype, similar to cCICs introduced to postnatal P3 heart with persistent cell cycle activity for up to 4 weeks. Fibroblast‐like phenotype of exogenously transferred cCICs in fetal and postnatal cardiogenic environments is consistent with inability to contribute directly toward cardiogenesis and lack of functional integration with host myocardium. In contrast, cCICs incorporation into extra‐embryonic membranes is consistent with fate of polyploid cells in blastocysts. These findings provide insight into cCIC biology, their inherent predisposition toward fibroblast fates in cardiogenic environments, and remarkable participation in extra‐embryonic tissue formation.

Automated segmentation of cardiomyocyte Z-disks from high-throughput scanning electron microscopy data

BackgroundWith the advent of new high-throughput electron microscopy techniques such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion-beam scanning electron microscopy (FIB-SEM) biomedical scientists can study sub-cellular structural mechanisms of heart disease at high resolution and high volume. Among several key components that determine healthy contractile function in cardiomyocytes are Z-disks or Z-lines, which are located at the lateral borders of the sarcomere, the fundamental unit of striated muscle. Z-disks play the important role of anchoring contractile proteins within the cell that make the heartbeat. Changes to their organization can affect the force with which the cardiomyocyte contracts and may also affect signaling pathways that regulate cardiomyocyte health and function. Compared to other components in the cell, such as mitochondria, Z-disks appear as very thin linear structures in microscopy data with limited difference in contrast to the remaining components of the cell.MethodsIn this paper, we propose to generate a 3D model of Z-disks within single adult cardiac cells from an automated segmentation of a large serial-block-face scanning electron microscopy (SBF-SEM) dataset. The proposed fully automated segmentation scheme is comprised of three main modules including “pre-processing”, “segmentation” and “refinement”. We represent a simple, yet effective model to perform segmentation and refinement steps. Contrast stretching, and Gaussian kernels are used to pre-process the dataset, and well-known “Sobel operators” are used in the segmentation module.ResultsWe have validated our model by comparing segmentation results with ground-truth annotated Z-disks in terms of pixel-wise accuracy. The results show that our model correctly detects Z-disks with 90.56% accuracy. We also compare and contrast the accuracy of the proposed algorithm in segmenting a FIB-SEM dataset against the accuracy of segmentations from a machine learning program called Ilastik and discuss the advantages and disadvantages that these two approaches have.ConclusionsOur validation results demonstrate the robustness and reliability of our algorithm and model both in terms of validation metrics and in terms of a comparison with a 3D visualisation of Z-disks obtained using immunofluorescence based confocal imaging.

P5382Geometry and strain sensing dictate YAP-dependent evolution of human cardiac stromal cells toward myofibroblasts in the cardiosphere organoid model

Abstract Background Cardiac stromal cells are the most abundant cell type present in the heart, where they have an important homeostatic function in matrix renewal and electromechanical coupling. We have shown that YAP transcription factor (a component of the Hippo pathway) is important for the proliferation of these cells and, potentially, pathologic cardiac matrix stiffening in heart failure. Purpose Given the role of Hippo pathway in cell mechanosensation, we assessed the conditions for activation of the YAP-dependent transcriptional pathway related to geometry/strain sensing using human “cardiosphere”-derived cells. Methods Human cardiospheres (C-Sp) and C-Sp-derived cells (CDCs) were obtained from fragments of atrial appendages of patients admitted for AoC bypass. C-Sps and CDCs were cultured using published methods. To analyze cell/nuclear geometry in 2D/3D culture, we employed an adapting thresholding algorithm allowing segmentation of cell membrane/nuclei shape and the relative abundance of nuclear/cytoplasmic fluorescence intensities from z-stacked confocal immunofluorescence images. Inhibitors of signaling cascades converging onto actin cytoskeleton polymerization or of YAP/TEADs interaction were used to assess YAP target genes transcription. Results Previous results showed stiffness sensitivity of YAP nuclear translocation process. Namely, CDCs plated onto stiff (E >50kPa) culture plates exhibited high ratios of nuclear/cytoplasmic YAP, as compared to cells plated on substrates with lower stiffness (E ∼ 10kPa). Analogously, cells with a stretched cytoplasm/nuclear shape in the border regions of the 3D organoids exhibited a higher YAP nuclear localization level and expression of fibrotic markers compared with cells with a more round geometry in the center of the spheres. To uncouple the cytoskeleton tensioning-dependent YAP nuclear shuttling, we treated CDCs plated onto stiff substrates with blebbistatin and Y27632. This showed a fully reversible YAP relocation from the nucleus to the cytoplasm and a reversion of canonical target genes (CYR61, ANKRD1 and CTGF) expression. In parallel, a “softening” process of the nucleus, likely due to release from cytoskeleton traction forces, was observed by confocal imaging along the cellular z-axis. Interestingly, use of Verteporfin, a drug directly interfering with the interaction of YAP/TAZ complex with TEADs DNA binding proteins showed reversion of pro-fibrotic YAP targets independent of high cytoskeleton tensioning. Conclusions These results establish, for the first time, a deterministic relationship between pro-pathologic evolution of human adult cardiac stromal cells and activation of mechanosensing-dependent pathways. They also provide quantitative criteria for interpreting fibrotic evolution of the heart based on modifications of myocardial architecture and mechanical compliance, as well as the ability of the cells to generate tension forces affecting the nuclear structure.

Abstract 257: Cortical Bone Stem Cell-Derived Exosomes Alter Wound Healing Response in Cardiac Fibroblasts and Cardiac Endothelial Cells

Rationale: Post-myocardial infarction (MI) results in remodeling that leads to the development of heart failure. We have previously seen improvements in post-MI wound healing and scar formation as a result of administration of cortical bone stem cell derived exosomes (CBSC exosomes). Objectives: We sought to further elucidate the mechanism through which CBSC exosomes improved scar formation and altered wound healing through in vitro experimentation. We continued to explore the unique characteristics of CBSC exosomes in order to understand why they have anti-fibrotic effects. Methods and Results: We performed migration assays on cardiac endothelial cells and cardiac fibroblasts, and saw alterations in cell migration in both cell types upon treatment with mCBSC CM and mCBSC exosomes. We discovered that migration of CECs was reduced by 20% under the influence of mCBSC CM and mCBSC exosomes, but rescued when exosomes were removed from mCBSC CM. We discovered that the removal of exosomes from mCBSC CM slowed cardiac fibroblast migration by 40%. We saw a decrease in the quantity of pro-fibrotic mRNA in cardiac endothelial cells and cardiac fibroblasts after treatment with mCBSC exosomes. Adult cardiac endothelial cell mRNA levels of VEGFA, Col4A1, and VCAM were reduced nearly two fold from control untreated cell levels following mCBSC exosome treatment. Adult cardiac fibroblast Col1A1, Col3A1, Col4A1, and MMP2 mRNA levels were reduced one-fold from control untreated cell levels following mCBSC exosome treatment. Conclusions: Our findings show that the wound healing induced by CBSC treatment post-MI involves a mechanism altering the migration of endothelial cells, sustaining the migratory capabilities of fibroblasts, and decreasing the production of pro-fibrotic mRNA in cardiac fibroblasts and cardiac endothelial cells.

Improved Protocol for Cardiac Differentiation and Maturation of Pluripotent Stem Cells.

Pluripotent stem cells feature the capacity to differentiate into any somatic cell types including cardiomyocytes. We report a cost-effective and simple protocol for the differentiation of specific ventricular cardiomyocytes. These cells are elongated, do not spontaneously beat, and do not feature any Ca2+-transient, an index of their stage of maturation toward adult cardiac cells. They represent a suitable model to screen both the efficiency and toxicology of drugs.

Apoptotic cell death in heart failure associated with diabetes

Diabetes is a serious public health problem. Improvements in the treatment of noncardiac complications from diabetes have resulted in heart disease becoming a leading cause of death in diabetic patients. Several cardiovascular pathological consequences of diabetes such as hypertension affect the heart to varying degrees. Furthermore, hyperglycemia as a sole risk factor may directly cause cardiovascular damage and lead to a complication such as diabetic cardiomyopathy. Alterations in myocardial structure and function occur during the late stage of diabetes. These chronic alterations are believed to result from acute cardiac responses to suddenly increased glucose levels at the early stage of diabetes. In failing hearts, cardiomyocytes degenerate and interstitial fibrosis, which indicates cardiomyocyte loss, becomes more prominent in the myocardium. Various mechanisms may establish an association of diabetes mellitus and heart failure as follows: first, comorbidities such as hypertension may play a role, diabetes also accelerates the development of coronary atherosclerosis, and experimental, clinical studies support the existence of microangiopathy, metabolic factors, or myocardial fibrosis influencing diabetic cardiomyopathy. Apoptosis of adult cardiac muscle cells can have lasting adverse consequences on overall cardiac performance. It is possible that autophagic degeneration may also be one of the mechanisms of myocardial cell death.

Abstract 470: Gata4, Mef2c and Tbx5 More Efficiently Transdifferentiate Endothelial Cells into Cardiomyocyte-like Cells Through Endothelial-Mesenchymal Transition Process

Background: The cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) have been used to successfully reprogram cardiac fibroblasts into induced cardiomyocyte-like cells (iCMs). However their effects on other cell types remain largely unknown. Endothelial cells are an important population of the heart by Pinto et al. (2016). Given the more profound iCM induction effects observed in vivo, we have speculated that reprogramming of endogenous endothelial cells may also play an important role. Purpose: To investigate the cellular reprograming effects and mechanisms of GMT factors on cardiac endothelial cells and compare the outcomes with that from cardiac fibroblasts. Methods: Adult rat cardiac endothelial cells (REC) and cardiac fibroblasts (RCF) were purchased commercially. The cells were cultured in appropriate media and infected with GMT-expressing lentivirus and maintained with iCM media. Fourteen days later Immunofluorescence staining, FACS, and qRT-PCR were performed. Results: Both GMT treated RCFs and RECs showed positive staining of cardiac marker cTnT and αActinin-positive cells via immunofluorescence staining. FACS analysis showed GMT treated RECs had 16.7% of cTnT positive cells versus 3.3% in GMT treated RCF’s. qRT-PCR results demonstrated significant up-regulation of cTnT in GMT infected RECs (74.4±23.3 times, p<0.05) as compared to the control group. Similar upregulation of cTnT was observed in RCFs but to a lesser degree (16.1±7.2 times, p=NS). Moreover, RECs presented a significant change in cell morphology from rounded cells in a cobblestone arrangement to elongated spindle-shaped cells during 14 days culture. In addition, qRT-PCR analysis demonstrated that endothelial-mesenchymal transition (EndoMT) markers such as Slug, Snail, Oct4, and αSMA were upregulated in the cells with GFP encoded virus infection and those markers were downregulated in the GMT-treated RECs (p<0.01 for Snail, p<0.05 for Slug, and αSMA). Conclusion: This report suggests that RECs are susceptive to GMT induced cardiac reprogramming than in RCFs. Our study also indicate that EMT and MET sequential process are involved in the reprogramming of RECs toward iCM cells. RECs could be targeted towards iCM generation for a higher reprogramming rate.

An automated workflow for segmenting single adult cardiac cells from large-volume serial block-face scanning electron microscopy data.

This paper presents a new algorithm to automatically segment the myofibrils, mitochondria and nuclei within single adult cardiac cells that are part of a large serial-block-face scanning electron microscopy (SBF-SEM) dataset. The algorithm only requires a set of manually drawn contours that roughly demarcate the cell boundary at routine slice intervals (every 50th, for example). The algorithm correctly classified pixels within the single cell with 97% accuracy when compared to manual segmentations. One entire cell and the partial volumes of two cells were segmented. Analysis of segmentations within these cells showed that myofibrils and mitochondria occupied 47.5% and 51.6% on average respectively, while the nuclei occupy 0.7% of the cell for which the entire volume was captured in the SBF-SEM dataset. Mitochondria clustering increased at the periphery of the nucleus region and branching points of the cardiac cell. The segmentations also showed high area fraction of mitochondria (up to 70% of the 2D image slice) in the sub-sarcolemmal region, whilst it was closer to 50% in the intermyofibrillar space. We finally demonstrate that our segmentations can be turned into 3D finite element meshes for cardiac cell computational physiology studies. We offer our large dataset and MATLAB implementation of the algorithm for research use at www.github.com/CellSMB/sbfsem-cardiac-cell-segmenter/. We anticipate that this timely tool will be of use to cardiac computational and experimental physiologists alike who study cardiac ultrastructure and its role in heart function.

An autofluorescence-based method for the isolation of highly purified ventricular cardiomyocytes.

The aim of our study was to set up a simple and reliable isolation method of living ventricular cardiomyocytes (vCMs) for molecular and biological studies. A standard technique for the retrograde perfusion of an enzymatic solution was used to isolate cardiac cells from adult mouse heart. Fluorescence-activated cell sorting (FACS) on adult murine cardiac ventricle cells was performed, comparing the intrinsic autofluorescence in the FITC channel and the forward scatter (FSC) parameter in order to isolate highly fluorescent cells. The expression of cell-specific mRNAs was assessed with real-time PCR in cells sorted on the basis of their FITC and FSC characteristics. We identified two distinct subpopulations of cells harvested after retrograde perfusion of wild-type heart: FITChigh/FSCdim and FITCdim/FSChigh. Immunophenotyping and mRNA analysis (qPCR and RNA sequencing) revealed that only FITChigh/FSCdim cells were highly enriched in CM markers. Genes with high expression in endothelial cells and fibroblasts were enriched in the FITCdim/FSChigh subpopulation. With the use of tdTomatofl/fl-α-myosin heavy chain MerCreMer+/-mouse heart, we found that tdTomato-positive vCMs were present in the FITChigh/FSCdim region but were only rare in the FITCdim/FSChigh fraction. We have developed a simple and reliable method for the isolation of highly purified vCMs from the adult murine myocardium, avoiding fixation and permeabilization steps. These isolated vCMs can be used in particular for detailed molecular studies, avoiding contamination with other myocardial cell types.