Spatially organized cellular communities form the developing human heart.

The heart, which is the first organ to develop, is highly dependent on its form to function1,2. However, how diverse cardiac cell types spatially coordinate to create the complex morphological structures that are crucial for heart function remains unclear. Here we integrated single-cell RNA-sequencing with high-resolution multiplexed error-robust fluorescence in situ hybridization to resolve the identity of the cardiac cell types that develop the human heart. This approach also provided a spatial mapping of individual cells that enables illumination of their organization into cellular communities that form distinct cardiac structures. We discovered that many of these cardiac cell types further specified into subpopulations exclusive to specific communities, which support their specialization according to the cellular ecosystem and anatomical region. In particular, ventricular cardiomyocyte subpopulations displayed an unexpected complex laminar organization across the ventricular wall and formed, with other cell subpopulations, several cellular communities. Interrogating cell-cell interactions within these communities using in vivo conditional genetic mouse models and in vitro human pluripotent stem cell systems revealed multicellular signalling pathways that orchestrate the spatial organization of cardiac cell subpopulations during ventricular wall morphogenesis. These detailed findings into the cellular social interactions and specialization of cardiac cell types constructing and remodelling the human heart offer new insights into structural heart diseases and the engineering of complex multicellular tissues for human heart repair.

Structure of the native myosin filament in the relaxed cardiac sarcomere.

The thick filament is a key component of sarcomeres, the basic units of striated muscle1. Alterations in thick filament proteins are associated with familial hypertrophic cardiomyopathy and other heart and muscle diseases2. Despite the central importance of the thick filament, its molecular organization remains unclear. Here we present the molecular architecture of native cardiac sarcomeres in the relaxed state, determined by cryo-electron tomography. Our reconstruction of the thick filament reveals the three-dimensional organization of myosin, titin and myosin-binding protein C (MyBP-C). The arrangement of myosin molecules is dependent on their position along the filament, suggesting specialized capacities in terms of strain susceptibility and force generation. Three pairs of titin-α and titin-ß chains run axially along the filament, intertwining with myosin tails and probably orchestrating the length-dependent activation of the sarcomere. Notably, whereas the three titin-α chains run along the entire length of the thick filament, titin-ß chains do not. The structure also demonstrates that MyBP-C bridges thin and thick filaments, with its carboxy-terminal region binding to the myosin tails and directly stabilizing the OFF state of the myosin heads in an unforeseen manner. These results provide a foundation for future research investigating muscle disorders involving sarcomeric components.

Urokinase-Type Plasminogen Activator Receptor Regulates Prosurvival and Angiogenic Properties of Cardiac Mesenchymal Stromal Cells.

One of the largest challenges to the implementation of cardiac cell therapy is identifying selective reparative targets to enhance stem/progenitor cell therapeutic efficacy. In this work, we hypothesized that such a target could be an urokinase-type plasminogen activator receptor (uPAR)-a glycosyl-phosphatidyl-inositol-anchored membrane protein, interacting with urokinase. uPAR is able to form complexes with various transmembrane proteins such as integrins, activating intracellular signaling pathway and thus regulating multiple cell functions. We focused on studying the CD117+ population of cardiac mesenchymal progenitor cells (MPCs), expressing uPAR on their surface. It was found that the number of CD117+ MPCs in the heart of the uPAR-/- mice is lower, as well as their ability to proliferate in vitro compared with cells from wild-type animals. Knockdown of uPAR in CD117+ MPCs of wild-type animals was accompanied by a decrease in survival rate and Akt signaling pathway activity and by an increase in the level of caspase activity in these cells. That suggests the role of uPAR in supporting cell survival. After intramyocardial transplantation of uPAR(-) MPCs, reduced cell retention and angiogenesis stimulation were observed in mice with myocardial infarction model compared to uPAR(+) cells transplantation. Taken together, the present results appear to prove a novel mechanism of uPAR action in maintaining the survival and angiogenic properties of CD117+ MPCs. These results emphasize the importance of the uPAR as a potential pharmacological target for the regulation of reparative properties of myocardial mesenchymal progenitor cells.

Spatially resolved multiomics of human cardiac niches.

The function of a cell is defined by its intrinsic characteristics and its niche: the tissue microenvironment in which it dwells. Here we combine single-cell and spatial transcriptomics data to discover cellular niches within eight regions of the human heart. We map cells to microanatomical locations and integrate knowledge-based and unsupervised structural annotations. We also profile the cells of the human cardiac conduction system1. The results revealed their distinctive repertoire of ion channels, G-protein-coupled receptors (GPCRs) and regulatory networks, and implicated FOXP2 in the pacemaker phenotype. We show that the sinoatrial node is compartmentalized, with a core of pacemaker cells, fibroblasts and glial cells supporting glutamatergic signalling. Using a custom CellPhoneDB.org module, we identify trans-synaptic pacemaker cell interactions with glia. We introduce a druggable target prediction tool, drug2cell, which leverages single-cell profiles and drug-target interactions to provide mechanistic insights into the chronotropic effects of drugs, including GLP-1 analogues. In the epicardium, we show enrichment of both IgG+ and IgA+ plasma cells forming immune niches that may contribute to infection defence. Overall, we provide new clarity to cardiac electro-anatomy and immunology, and our suite of computational approaches can be applied to other tissues and organs.

Metabolic landscape in cardiac aging: insights into molecular biology and therapeutic implications.

Cardiac aging is evident by a reduction in function which subsequently contributes to heart failure. The metabolic microenvironment has been identified as a hallmark of malignancy, but recent studies have shed light on its role in cardiovascular diseases (CVDs). Various metabolic pathways in cardiomyocytes and noncardiomyocytes determine cellular senescence in the aging heart. Metabolic alteration is a common process throughout cardiac degeneration. Importantly, the involvement of cellular senescence in cardiac injuries, including heart failure and myocardial ischemia and infarction, has been reported. However, metabolic complexity among human aging hearts hinders the development of strategies that targets metabolic susceptibility. Advances over the past decade have linked cellular senescence and function with their metabolic reprogramming pathway in cardiac aging, including autophagy, oxidative stress, epigenetic modifications, chronic inflammation, and myocyte systolic phenotype regulation. In addition, metabolic status is involved in crucial aspects of myocardial biology, from fibrosis to hypertrophy and chronic inflammation. However, further elucidation of the metabolism involvement in cardiac degeneration is still needed. Thus, deciphering the mechanisms underlying how metabolic reprogramming impacts cardiac aging is thought to contribute to the novel interventions to protect or even restore cardiac function in aging hearts. Here, we summarize emerging concepts about metabolic landscapes of cardiac aging, with specific focuses on why metabolic profile alters during cardiac degeneration and how we could utilize the current knowledge to improve the management of cardiac aging.

Gßγ subunits colocalize with RNA polymerase II and regulate transcription in cardiac fibroblasts.

Gßγ subunits mediate many different signaling processes in various compartments of the cell, including the nucleus. To gain insight into the functions of nuclear Gßγ signaling, we investigated the functional role of Gßγ signaling in the regulation of GPCR-mediated gene expression in primary rat neonatal cardiac fibroblasts. We identified a novel, negative, regulatory role for the Gß1γ dimer in the fibrotic response. Depletion of Gß1 led to derepression of the fibrotic response at the mRNA and protein levels under basal conditions and an enhanced fibrotic response after sustained stimulation of the angiotensin II type I receptor. Our genome-wide chromatin immunoprecipitation experiments revealed that Gß1 colocalized and interacted with RNA polymerase II on fibrotic genes in an angiotensin II-dependent manner. Additionally, blocking transcription with inhibitors of Cdk9 prevented association of Gßγ with transcription complexes. Together, our findings suggest that Gß1γ is a novel transcriptional regulator of the fibrotic response that may act to restrict fibrosis to conditions of sustained fibrotic signaling. Our work expands the role for Gßγ signaling in cardiac fibrosis and may have broad implications for the role of nuclear Gßγ signaling in other cell types.

Lineage tracing clarifies the cellular origin of tissue-resident macrophages in the developing heart.

Tissue-resident macrophages play essential functions in the maintenance of tissue homeostasis and repair. Recently, the endocardium has been reported as a de novo hemogenic site for the contribution of hematopoietic cells, including cardiac macrophages, during embryogenesis. These observations challenge the current consensus that hematopoiesis originates from the hemogenic endothelium within the yolk sac and dorsal aorta. Whether the developing endocardium has such a hemogenic potential requires further investigation. Here, we generated new genetic tools to trace endocardial cells and reassessed their potential contribution to hematopoietic cells in the developing heart. Fate-mapping analyses revealed that the endocardium contributed minimally to cardiac macrophages and circulating blood cells. Instead, cardiac macrophages were mainly derived from the endothelium during primitive/transient definitive (yolk sac) and definitive (dorsal aorta) hematopoiesis. Our findings refute the concept of endocardial hematopoiesis, suggesting that the developing endocardium gives rise minimally to hematopoietic cells, including cardiac macrophages.

Suppression of the Inhibitory Effect of circ_0036176-Translated Myo9a-208 on Cardiac Fibroblast Proliferation by miR-218-5p.

Increasing evidence has shown that circular RNAs (circRNAs) participate in the process of cardiac remodeling. CircRNA circ_0036176 originating from the back-splicing of exon 2 to exon4 of myosin IXA (Myo9a) gene was shown to be increased in the myocardium of patients with heart failure (HF) and riched in exosomes from human AC16 cardiomyocytes with overexpression of circ_0036176. Proliferation activity was inhibited in mCFs subjected to exosomal circ_0036176 treatment and in mCFs with overexpression of circ_0036176. Interestingly, circ_0036176 contains an IRES element and an ORF of 627 nt encoding a 208-amino acid protein (termed as Myo9a-208). Myo9a-208 was shown to mediate the inhibitory effect of circ_0036176 on CFs proliferation, and miR-218-5p could inhibit Myo9a-208 expression by binding to circ_0036176, resulting in abolishing the effect of circ_0036176 on inactivating cyclin/Rb signal and suppressing CFs proliferation. Our findings suggest that circ_0036176 inhibits mCFs proliferation by translating Myo9a-208 protein to suppress cyclin/Rb pathway.

Heart regeneration: 20 years of progress and renewed optimism.

Cardiovascular disease is a leading cause of death worldwide, and thus there remains great interest in regenerative approaches to treat heart failure. In the past 20 years, the field of heart regeneration has entered a renaissance period with remarkable progress in the understanding of endogenous heart regeneration, stem cell differentiation for exogenous cell therapy, and cell-delivery methods. In this review, we highlight how this new understanding can lead to viable strategies for human therapy. For the near term, drugs, electrical and mechanical devices, and heart transplantation will remain mainstays of cardiac therapies, but eventually regenerative therapies based on fundamental regenerative biology may offer more permanent solutions for patients with heart failure.

Sphingosine 1-Phosphate-Upregulated COX-2/PGE2 System Contributes to Human Cardiac Fibroblast Apoptosis: Involvement of MMP-9-Dependent Transactivation of EGFR Cascade.

Human cardiac fibroblasts (HCFs) play key roles in normal physiological functions and pathological processes in the heart. Our recent study has found that, in HCFs, sphingosine 1-phosphate (S1P) can upregulate the expression of cyclooxygenase-2 (COX-2) leading to prostaglandin E2 (PGE2) generation mediated by S1P receptors/PKCα/MAPKs cascade-dependent activation of NF-κB. Alternatively, G protein-coupled receptor- (GPCR-) mediated transactivation of receptor tyrosine kinases (RTKs) has been proved to induce inflammatory responses. However, whether GPCR-mediated transactivation of RTKs participated in the COX-2/PGE2 system induced by S1P is still unclear in HCFs. We hypothesize that GPCR-mediated transactivation of RTKs-dependent signaling cascade is involved in S1P-induced responses. This study is aimed at exploring the comprehensive mechanisms of S1P-promoted COX-2/PGE2 expression and apoptotic effects on HCFs. Here, we used pharmacological inhibitors and transfection with siRNA to evaluate whether matrix metalloprotease (MMP)2/9, heparin-binding- (HB-) epidermal growth factor (EGF), EGF receptor (EGFR), PI3K/Akt, MAPKs, and transcription factor AP-1 participated in the S1P-induced COX-2/PGE2 system determined by Western blotting, real-time polymerase chain reaction (RT-PCR), chromatin immunoprecipitation (ChIP), and promoter-reporter assays in HCFs. Our results showed that S1PR1/3 activated by S1P coupled to Gq- and Gi-mediated MMP9 activity to stimulate EGFR/PI3K/Akt/MAPKs/AP-1-dependent activity of transcription to upregulate COX-2 accompanied with PGE2 production, leading to stimulation of caspase-3 activity and apoptosis. Moreover, S1P-enhanced c-Jun bound to COX-2 promoters on its corresponding binding sites, which was attenuated by these inhibitors of protein kinases, determined by a ChIP assay. These results concluded that transactivation of MMP9/EGFR-mediated PI3K/Akt/MAPKs-dependent AP-1 activity was involved in the upregulation of the COX-2/PGE2 system induced by S1P, in turn leading to apoptosis in HCFs.

Hdac8 Inhibitor Alleviates Transverse Aortic Constriction-Induced Heart Failure in Mice by Downregulating Ace1.

BACKGROUND: Heart failure is characterized by activation of the renin-angiotensin-aldosterone system, which is involved in the regulation of cardiac hypertrophy and hypertension. Recently, we reported that Hdac8 inhibition alleviates isoproterenol-induced and angiotensin II-induced cardiac hypertrophy or hypertension in mice. Here, the effect and regulatory mechanisms of the Hdac8 selective inhibitor PCI34051 on pressure overload-induced heart failure were examined. METHODS AND RESULTS: At week 6 posttransverse aortic constriction (TAC), mice were administered with PCI34051 (3, 10, or 30 mg/kg bodyweight/day) for 2 weeks. The therapeutic effects of PCI34051 on TAC-induced cardiac and lung hypertrophy were determined by examining the heart weight-to-bodyweight and lung weight-to-bodyweight ratios and the cross-sectional cardiomyocyte area. Echocardiography analysis revealed that PCI34051 mitigated TAC-induced decreased ejection fraction and fractional shortening. Additionally, the expression of Hdac8 was upregulated in the cardiac and pulmonary tissues of TAC mice. The expression levels of Ace1 and Agtr1 were upregulated, whereas those of Ace2 and Agtr2 were downregulated in TAC mice. PCI34051 treatment or Hdac8 knockdown alleviated inflammation as evidenced by Rela downregulation and Nfkbia upregulation in mice, as well as in cardiomyocytes, but not in cardiac fibroblasts. Hdac8 overexpression-induced Rela pathway activation was downregulated in Ace1 knockdown cells. Picrosirius red staining, real-time polymerase chain reaction, and western blotting analyses revealed that PCI34051 alleviated fibrosis and downregulated fibrosis-related genes. Moreover, PCI34051 or Hdac8 knockdown in rat cardiac fibroblasts alleviated cardiac fibrosis through the Tgfb1-Smad2/3 pathway. The results of overexpression and knockdown experiments revealed that Hdac8 and Ace1 promote inflammation and fibrosis. CONCLUSIONS: Treatment with PCI34051 enhanced cardiac and lung functions in the TAC-induced heart failure mouse model. These data suggest that HDAC8 is a potential novel therapeutic target for heart failure accompanied by pathological lung diseases.

Zebrafish integrin a3b is required for cardiac contractility and cardiomyocyte proliferation.

In cardiac muscle cells, heterodimeric integrin transmembrane receptors are known to serve as mechanotransducers, translating mechanical force to biochemical signaling. However, the roles of many individual integrins have still not been delineated. In this report, we demonstrate that Itga3b is localized to the sarcolemma of cardiomyocytes from 24 to 96 hpf. We further show that heterozygous and homozygous itga3b/bdf mutant embryos display a cardiomyopathy phenotype, with decreased cardiac contractility and reduced cardiomyocyte number. Correspondingly, proliferation of ventricular and atrial cardiomyoctyes and ventricular epicardial cells is decreased in itga3b mutant hearts. The contractile dysfunction of itga3b mutants can be attributed to cardiomyocyte sarcomeric disorganization, including thin myofilaments with blurred and shortened Z-discs. Together, our results reveal that Itga3b localizes to the myocardium sarcolemma, and it is required for cardiac contractility and cardiomyocyte proliferation.

Fibroblast mechanotransduction network predicts targets for mechano-adaptive infarct therapies.

Regional control of fibrosis after myocardial infarction is critical for maintaining structural integrity in the infarct while preventing collagen accumulation in non-infarcted areas. Cardiac fibroblasts modulate matrix turnover in response to biochemical and biomechanical cues, but the complex interactions between signaling pathways confound efforts to develop therapies for regional scar formation. We employed a logic-based ordinary differential equation model of fibroblast mechano-chemo signal transduction to predict matrix protein expression in response to canonical biochemical stimuli and mechanical tension. Functional analysis of mechano-chemo interactions showed extensive pathway crosstalk with tension amplifying, dampening, or reversing responses to biochemical stimuli. Comprehensive drug target screens identified 13 mechano-adaptive therapies that promote matrix accumulation in regions where it is needed and reduce matrix levels in regions where it is not needed. Our predictions suggest that mechano-chemo interactions likely mediate cell behavior across many tissues and demonstrate the utility of multi-pathway signaling networks in discovering therapies for context-specific disease states.

Monitoring the maturation of the sarcomere network: a super-resolution microscopy-based approach.

The in vitro generation of human cardiomyocytes derived from induced pluripotent stem cells (iPSC) is of great importance for cardiac disease modeling, drug-testing applications and for regenerative medicine. Despite the development of various cultivation strategies, a sufficiently high degree of maturation is still a decisive limiting factor for the successful application of these cardiac cells. The maturation process includes, among others, the proper formation of sarcomere structures, mediating the contraction of cardiomyocytes. To precisely monitor the maturation of the contractile machinery, we have established an imaging-based strategy that allows quantitative evaluation of important parameters, defining the quality of the sarcomere network. iPSC-derived cardiomyocytes were subjected to different culture conditions to improve sarcomere formation, including prolonged cultivation time and micro patterned surfaces. Fluorescent images of α-actinin were acquired using super-resolution microscopy. Subsequently, we determined cell morphology, sarcomere density, filament alignment, z-Disc thickness and sarcomere length of iPSC-derived cardiomyocytes. Cells from adult and neonatal heart tissue served as control. Our image analysis revealed a profound effect on sarcomere content and filament orientation when iPSC-derived cardiomyocytes were cultured on structured, line-shaped surfaces. Similarly, prolonged cultivation time had a beneficial effect on the structural maturation, leading to a more adult-like phenotype. Automatic evaluation of the sarcomere filaments by machine learning validated our data. Moreover, we successfully transferred this approach to skeletal muscle cells, showing an improved sarcomere formation cells over different differentiation periods. Overall, our image-based workflow can be used as a straight-forward tool to quantitatively estimate the structural maturation of contractile cells. As such, it can support the establishment of novel differentiation protocols to enhance sarcomere formation and maturity.

Twist regulates Yorkie activity to guide lineage reprogramming of syncytial alary muscles.

Genesis of syncytial muscles is typically considered as a paradigm for an irreversible developmental process. Notably, transdifferentiation of syncytial muscles is naturally occurring during Drosophila development. The ventral longitudinal heart-associated musculature (VLM) arises by a unique mechanism that revokes differentiation states of so-called alary muscles and comprises at least two distinct steps: syncytial muscle cell fragmentation into single myoblasts and successive reprogramming into founder cells that orchestrate de novo fiber formation of the VLM lineage. Here, we provide evidence that the mesodermal master regulator twist plays a key role during this reprogramming process. Acting downstream of Drosophila Tbx1 (Org-1), Twist is regulating the activity of the Hippo pathway effector Yorkie and is required for the initiation of syncytial muscle dedifferentiation and fragmentation. Subsequently, fibroblast growth factor receptor (FGFR)-Ras-mitogen-activated protein kinase (MAPK) signaling in resulting mononucleated myoblasts maintains Twist expression, thereby stabilizing nuclear Yorkie activity and inducing their lineage switch into founder cells of the VLM.

Biodistribution of unmodified cardiosphere-derived cell extracellular vesicles using single RNA tracing.

Extracellular vesicles (EVs) are potent signalling mediators. Although interest in EV translation is ever-increasing, development efforts are hampered by the inability to reliably assess the uptake of EVs and their RNA cargo. Here, we establish a novel qPCR-based method for the detection of unmodified EVS using an RNA Tracer (DUST). In this proof-of-concept study we use a human-specific Y RNA-derived small RNA (YsRNA) we dub "NT4" that is enriched in cardiosphere-derived cell small EVs (CDC-sEVs). The assay is robust, sensitive, and reproducible. Intravenously administered CDC-sEVs accumulated primarily in the heart on a per mg basis. Cardiac injury enhanced EV uptake in the heart, liver, and brain. Inhibition of EV docking by heparin suppressed uptake variably, while inhibition of endocytosis attenuated uptake in all organs. In vitro, EVs were uptaken more efficiently by macrophages, endothelial cells, and cardiac fibroblasts compared to cardiomyocytes. These findings demonstrate the utility of DUST to assess uptake of EVs in vivo and in vitro.

miR-30a-5p inhibits the proliferation and collagen formation of cardiac fibroblasts in diabetic cardiomyopathy.

Cardiac fibrosis is one of the major pathological characteristics of diabetic cardiomyopathy (DCM). MicroRNAs (miRNAs, miRs) have been identified as key regulators in the progression of cardiac fibrosis. This study aimed to investigate the role of miR-30a-5p in DCM and the underlying mechanism. The rat model of diabetes mellitus (DM) was established by streptozotocin injection, and the rat primary cardiac fibroblasts (CFs) were isolated from cardiac tissue and then treated with high glucose (HG). MTT assay was performed to assess the viability of CFs. Dual-luciferase reporter gene assay was conducted to verify the interaction between miR-30a-5p and Smad2. The expression of miR-30a-5p was downregulated in the myocardial tissues of DM rats and HG-stimulated CFs. Overexpression of miR-30a-5p reduced Smad2 levels and inhibited collagen formation in HG-stimulated CFs and DM rats, as well as decreased the proliferation of CFs induced by HG. Smad2 was a target of miR-30a-5p and its expression was inhibited by miR-30a-5p. Furthermore, the simultaneous overexpression of Smad2 and miR-30a-5p reversed the effect of miR-30a-5p overexpression alone in CFs. Our results indicated that miR-30a-5p reduced Smad2 expression and also induced a decrease in proliferation and collagen formation in DCM.

SEPHS1 is dispensable for pluripotency maintenance but indispensable for cardiac differentiation in mouse embryonic stem cells.

Embryonic stem cells (ESCs) are derived from the inner cell mass of developing blastocysts, which have self-renewal ability and have the potential to develop or reconstitute into all embryonic lineages. Selenophosphate synthetase 1 (SEPHS1) is an essential protein in mouse early embryo development. However, the role of SEPHS1 in mouse ESCs remains to be elucidated. In this study, we generated Sephs1 KO ESCs and found that deficiency of SEPSH1 has little effect on pluripotency maintenance and proliferation. Notably, SEPHS1 deficiency impaired differentiation into three germ layers and gastruloid aggregation in vitro. RNA-seq analysis showed SEPHS1 is involved in cardiogenesis, verified by no beating signal in Sephs1 KO embryoid body at d10 and low expression of cardiac-related and contraction markers. Taken together, our results suggest that SPEHS1 is dispensable in ESC self-renewal, but indispensable in subsequent germ layer differentiation especially for functional cardiac lineage.

Isolation methods of large and small extracellular vesicles derived from cardiovascular progenitors: A comparative study.

Since the discovery of the beneficial therapeutical effects of extracellular vesicles (EVs), these agents have been attracting great interest as next-generation therapies. EVs are nanosized membrane bodies secreted by all types of cells that mediate cell-cell communication. Although the classification of different subpopulations of EVs can be complex, they are broadly divided into microvesicles and exosomes based on their biogenesis and in large and small EVs based on their size. As this is an emerging field, current investigations are focused on basic aspects such as the more convenient method for EV isolation. In the present paper, we used cardiac progenitor cells (CPCs) to study and compare different cell culture conditions for EV isolation as well as two of the most commonly employed purification methods: ultracentrifugation (UC) and size-exclusion chromatography (SEC). Large and small EVs were separately analysed. We found that serum starvation of cells during the EV collecting period led to a dramatic decrease in EV secretion and major cell death. Regarding the isolation method, our findings suggest that UC and SEC gave similar EV recovery rates. Separation of large and small EV-enriched subpopulations was efficiently achieved with both purification protocols although certain difference in sample heterogeneity was observed. Noteworthy, while calnexin was abundant in large EVs, ALIX and CD63 were mainly found in small EVs. Finally, when the functionality of EVs was assessed on primary culture of adult murine cardiac fibroblasts, we found that EVs were taken up by these cells, which resulted in a pronounced reduction in the proliferative and migratory capacity of the cells. Specifically, a tendency towards a larger effect of SEC-related EVs was observed. No differences could be found between large and small EVs. Altogether, these results contribute to establish the basis for the use of EVs as therapeutic platforms, in particular in regenerative fields.

Long Noncoding RNAs Testis Development Related Gene 1 Aggravates Transforming Growth Factor-ß1-Induced Fibrogenesis and Inflammatory Response of Cardiac Fibroblasts Via miR-605-3p/Tumor Necrosis Factor Receptor Superfamily-21 Axis.

ABSTRACT: Heart failure is mainly caused by a decline in the systolic function of the heart. Long noncoding RNAs are related to cardiac diseases. This study aimed to explore the effects of long noncoding RNAs testis development related gene 1 (TDRG1) on the fibrogenesis and inflammatory response of transforming growth factor-beta1 (TGF-ß1)-stimulated human cardiac fibroblasts (HCFs). Levels of proinflammatory cytokines were evaluated by enzyme-linked immunosorbent assay. Reverse-transcription quantitative polymerase chain reaction was applied to reveal the expression levels of TDRG1, miR-605-3p, and tumor necrosis factor receptor superfamily (TNFRSF21). Western blot analysis was prepared to detect protein levels of TNFRSF21 and fibrosis-related genes. Luciferase reporter assay was conducted for confirming the interaction between miR-605-3p and TDRG1/TNFRSF21. We found that TGF-ß1-stimulated HCFs showed high concentrations of proinflammatory cytokines and increased protein levels of fibrosis-related genes, suggesting the dysfunctions of TGF-ß1-stimulated HCFs. In addition, TDRG1 was upregulated in TGF-ß1-stimulated HCFs. We found that interfering with TDRG1 alleviated dysfunctions of TGF-ß1-stimulated HCFs. Moreover, TDRG1 bound with miR-605-3p. MiR-605-3p exerted the antifibrogenic and anti-inflammatory effects in TGF-ß1-treated HCFs. As a target gene of miR-605-3p, TNFRSF21 reversed the antifibrogenic and anti-inflammatory effects of TDRG1 knockdown in TGF-ß1-treated HCFs. Overall, our study confirmed that TDRG1 aggravates fibrogenesis and inflammatory response in TGF-ß1-treated HCFs via the miR-605-3p/TNFRSF21 axis.