Expression Of Cardiac Markers Research Articles

APLNR marks a cardiac progenitor derived with human induced pluripotent stem cells

Cardiomyocytes can be readily derived from human induced pluripotent stem cell (hiPSC) lines, yet its efficacy varies across different batches of the same and different hiPSC lines. To unravel the inconsistencies of in vitro cardiac differentiation, we utilized single cell transcriptomics on hiPSCs undergoing cardiac differentiation and identified cardiac and extra-cardiac lineages throughout differentiation. We further identified APLNR as a surface marker for in vitro cardiac progenitors and immunomagnetically isolated them. Differentiation of isolated in vitro APLNR+ cardiac progenitors derived from multiple hiPSC lines resulted in predominantly cardiomyocytes accompanied with cardiac mesenchyme. Transcriptomic analysis of differentiating in vitro APLNR+ cardiac progenitors revealed transient expression of cardiac progenitor markers before further commitment into cardiomyocyte and cardiac mesenchyme. Analysis of in vivo human and mouse embryo single cell transcriptomic datasets have identified APLNR expression in early cardiac progenitors of multiple lineages. This platform enables generation of in vitro cardiac progenitors from multiple hiPSC lines without genetic manipulation, which has potential applications in studying cardiac development, disease modelling and cardiac regeneration.

Conductive Bacterial Nanocellulose-Polypyrrole Patches Promote Cardiomyocyte Differentiation.

The low endogenous regenerative capacity of the heart, added to the prevalence of cardiovascular diseases, triggered the advent of cardiac tissue engineering in the last decades. The myocardial niche plays a critical role in directing the function and fate of cardiomyocytes; therefore, engineering a biomimetic scaffold holds excellent promise. We produced an electroconductive cardiac patch of bacterial nanocellulose (BC) with polypyrrole nanoparticles (Ppy NPs) to mimic the natural myocardial microenvironment. BC offers a 3D interconnected fiber structure with high flexibility, which is ideal for hosting Ppy nanoparticles. BC-Ppy composites were produced by decorating the network of BC fibers (65 ± 12 nm) with conductive Ppy nanoparticles (83 ± 8 nm). Ppy NPs effectively augment the conductivity, surface roughness, and thickness of BC composites despite reducing scaffolds' transparency. BC-Ppy composites were flexible (up to 10 mM Ppy), maintained their intricate 3D extracellular matrix-like mesh structure in all Ppy concentrations tested, and displayed electrical conductivities in the range of native cardiac tissue. Furthermore, these materials exhibit tensile strength, surface roughness, and wettability values appropriate for their final use as cardiac patches. In vitro experiments with cardiac fibroblasts and H9c2 cells confirmed the exceptional biocompatibility of BC-Ppy composites. BC-Ppy scaffolds improved cell viability and attachment, promoting a desirable cardiomyoblast morphology. Biochemical analyses revealed that H9c2 cells showed different cardiomyocyte phenotypes and distinct levels of maturity depending on the amount of Ppy in the substrate used. Specifically, the employment of BC-Ppy composites drives partial H9c2 differentiation toward a cardiomyocyte-like phenotype. The scaffolds increase the expression of functional cardiac markers in H9c2 cells, indicative of a higher differentiation efficiency, which is not observed with plain BC. Our results highlight the remarkable potential use of BC-Ppy scaffolds as a cardiac patch in tissue regenerative therapies.

6-Formylindolo[3,2-b]carbazole Protects Against Angiotensin II-Induced Cellular Hypertrophy through the Induction of Cytochrome P450 1A1 and Its Associated 19(S)-HETE Metabolite In Vitro.

Aryl hydrocarbon receptor (AhR) is a multifunctional receptor that regulates cytochrome P450 1A1 (CYP1A1), an arachidonic acid (AA) metabolizing enzyme producing 19-hydroxyeicosatetraenoic acid (HETE). 6-formylindolo[3,2-b]carbazole (FICZ) demonstrates great affinity toward the AhR. Recently, we have shown that 19(S)-HETE is preferentially cardioprotective. This study investigates the role of FICZ on AhR and cytochrome P450 (CYP) 1A1-mediated AA metabolism and whether it attenuates angiotensin (Ang) II-induced cardiac hypertrophy. Adult human ventricular cardiomyocytes cell line treated with FICZ in the presence and absence of Ang II 10 μM. Protein levels of AhR and CYPs were determined by Western blot analysis and the mRNA expression of cardiac hypertrophic markers and CYPs were determined by real-time polymerase chain reaction. CYP1A1 enzyme activity and proteasomal degradation were determined by 7-ethoxyresorufin O-deethylase and proteasome 20S activity assays, respectively. Liquid chromatography tandem mass spectrometry was used to measure AA metabolites. Our results show that Ang II-induced cardiac hypertrophy modulates AA metabolites in an enantioselective manner, and that FICZ activates AhR in a time-dependent manner, inhibits AhR proteasomal degradation, induces CYP1A1, increases the concentration of 19(S)-HETE, and attenuates Ang II-induced cardiac hypertrophy by inhibiting the hypertrophic markers and decreasing cell surface area through midchain-HETE-dependent mechanism. In conclusion, the results demonstrate the ability of FICZ to protect against Ang II-induced cardiac hypertrophy by increasing the concentration of 19(S)-HETE through AhR regulated enzyme induction and inhibition of midchain-HETEs metabolites. SIGNIFICANCE STATEMENT: This study shows that 6-formylindolo[3,2-b]carbazole attenuate angiotensin II-induced cellular hypertrophy. The novel findings of our investigation are in characterizing the aryl hydrocarbon receptor involvement and the enantioselective differences in arachidonic acid metabolism in cardiac hypertrophy, which opens a new pathway to tackle and eventually treat heart failure.

Amniotic Fluid Mesenchymal Stromal Cells Derived from Fetuses with Isolated Cardiac Defects Exhibit Decreased Proliferation and Cardiomyogenic Potential.

Amniotic fluid mesenchymal stromal cells (AF-MSCs) represent an autologous cell source to ameliorate congenital heart defects (CHDs) in children. The AF-MSCs, having cardiomyogenic potential and being of fetal origin, may reflect the physiological and pathological changes in the fetal heart during embryogenesis. Hence, the study of defects in the functional properties of these stem cells during fetal heart development will help obtain a better understanding of the cause of neonatal CHDs. Therefore, in the present study, we compared the proliferative and cardiomyogenic potential of AF-MSCs derived from ICHD fetuses (ICHD AF-MSCs) with AF-MSCs from structurally normal fetuses (normal AF-MSCs). Compared to normal AF-MSCs, the ICHD AF-MSCs showed comparable immunophenotypic MSC marker expression and adipogenic and chondrogenic differentiation potential, with decreased proliferation, higher senescence, increased expression of DNA-damaged genes, and osteogenic differentiation potential. Furthermore, the expression of cardiac progenitor markers (PDGFR-α, VEGFR-2, and SSEA-1), cardiac transcription factors (GATA-4, NKx 2-5, ISL-1, TBX-5, TBX-18, and MeF-2C), and cardiovascular markers (cTNT, CD31, and α-SMA) were significantly reduced in ICHD AF-MSCs. Overall, these results suggest that the AF-MSCs of ICHD fetuses have proliferation defects with significantly decreased cardiomyogenic differentiation potential. Thus, these defects in ICHD AF-MSCs highlight that the impaired heart development in ICHD fetuses may be due to defects in the stem cells associated with heart development during embryogenesis.

Dose-dependent Effects of PRC2 and HDAC Inhibitors on Cardiomyocyte Hypertrophy Induced by Phenylephrine.

Postnatal cardiomyocytes respond to stress signals by hypertrophic growth and fetal gene reprogramming, which involves epigenetic remodeling mediated by histone methyltransferase polycomb repressive complex 2 (PRC2) and histone deacetylases (HDACs). However, it remains unclear to what extent these histone modifiers contribute to the development of cardiomyocyte hypertrophy. Neonatal rat ventricular myocytes (NRVMs) were stimulated by phenylephrine (PE; 50μM) to induce hypertrophy in the presence or absence of the PRC2 inhibitor GSK126 or the HDACs inhibitor Trichostatin A (TSA). Histone methylation and acetylation were measured by Western blot. Cell size was determined by wheat germ agglutinin (WGA) staining. Cardiac hypertrophy markers were quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). PE treatment induced the expression of cardiac hypertrophy markers, including natriuretic peptide A (Nppa), natriuretic peptide B (Nppb), and myosin heavy chain 7 (Myh7), in a time-dependent manner in NRVMs. Histone modifications, including H3K27me3, H3K9ac, and H3K27ac, were dynamically altered after PE treatment. Treatment with TSA and GSK126 dose-dependently repressed histone acetylation and methylation, respectively. While TSA reversed the PE-induced cell size enlargement in a wide range of concentrations, cardiomyocyte hypertrophy was only inhibited by GSK126 at a higher dose (1μM). Consistently, TSA dose-dependently suppressed the induction of Nppa, Nppb, and Myh7/Myh6 ratio, while these indexes were only inhibited by GSK126 at 1μM. However, TSA, but not GSK126, caused pro-hypertrophic expression of pathological genes at the basal level. Our data demonstrate diversified effects of TSA and GSK126 on PE-induced cardiomyocyte hypertrophy, and shed light on epigenetic reprogramming in the pathogenesis of cardiac hypertrophy.

Gene-Edited Human-Induced Pluripotent Stem Cell Lines to Elucidate DAND5 Function throughout Cardiac Differentiation.

(1) Background: The contribution of gene-specific variants for congenital heart disease, one of the most common congenital disabilities, is still far from our complete understanding. Here, we applied a disease model using human-induced pluripotent stem cells (hiPSCs) to evaluate the function of DAND5 on human cardiomyocyte (CM) differentiation and proliferation. (2) Methods: Taking advantage of our DAND5 patient-derived iPSC line, we used CRISPR-Cas9 gene-editing to generate a set of isogenic hiPSCs (DAND5-corrected and DAND5 full-mutant). The hiPSCs were differentiated into CMs, and RT-qPCR and immunofluorescence profiled the expression of cardiac markers. Cardiomyocyte proliferation was analysed by flow cytometry. Furthermore, we used a multi-electrode array (MEA) to study the functional electrophysiology of DAND5 hiPSC-CMs. (3) Results: The results indicated that hiPSC-CM proliferation is affected by DAND5 levels. Cardiomyocytes derived from a DAND5 full-mutant hiPSC line are more proliferative when compared with gene-corrected hiPSC-CMs. Moreover, parallel cardiac differentiations showed a differential cardiac gene expression profile, with upregulated cardiac progenitor markers in DAND5-KO hiPSC-CMs. Microelectrode array (MEA) measurements demonstrated that DAND5-KO hiPSC-CMs showed prolonged field potential duration and increased spontaneous beating rates. In addition, conduction velocity is reduced in the monolayers of hiPSC-CMs with full-mutant genotype. (4) Conclusions: The absence of DAND5 sustains the proliferation of hiPSC-CMs, which alters their electrophysiological maturation properties. These results using DAND5 hiPSC-CMs consolidate the findings of the in vitro and in vivo mouse models, now in a translational perspective. Altogether, the data will help elucidate the molecular mechanism underlying this human heart disease and potentiates new therapies for treating adult CHD.

Dysregulated Cell Homeostasis and miRNAs in Human iPSC-Derived Cardiomyocytes from a Propionic Acidemia Patient with Cardiomyopathy.

Propionic acidemia (PA) disorder shows major involvement of the heart, among other alterations. A significant number of PA patients develop cardiac complications, and available evidence suggests that this cardiac dysfunction is driven mainly by the accumulation of toxic metabolites. To contribute to the elucidation of the mechanistic basis underlying this dysfunction, we have successfully generated cardiomyocytes through the differentiation of induced pluripotent stem cells (iPSCs) from a PCCB patient and its isogenic control. In this human cellular model, we aimed to examine microRNAs (miRNAs) profiles and analyze several cellular pathways to determine miRNAs activity patterns associated with PA cardiac phenotypes. We have identified a series of upregulated cardiac-enriched miRNAs and alterations in some of their regulated signaling pathways, including an increase in the expression of cardiac damage markers and cardiac channels, an increase in oxidative stress, a decrease in mitochondrial respiration and autophagy; and lipid accumulation. Our findings indicate that miRNA activity patterns from PA iPSC-derived cardiomyocytes are biologically informative and advance the understanding of the molecular mechanisms of this rare disease, providing a basis for identifying new therapeutic targets for intervention strategies.

Three-dimensional heart extracellular matrix enhances chemically induced direct cardiac reprogramming

Direct cardiac reprogramming has emerged as a promising therapeutic approach for cardiac regeneration. Full chemical reprogramming with small molecules to generate cardiomyocytes may be more amenable than genetic reprogramming for clinical applications as it avoids safety concerns associated with genetic manipulations. However, challenges remain regarding low conversion efficiency and incomplete cardiomyocyte maturation. Furthermore, the therapeutic potential of chemically induced cardiomyocytes (CiCMs) has not been investigated. Here, we report that a three-dimensional microenvironment reconstituted with decellularized heart extracellular matrix can enhance chemical reprogramming and cardiac maturation of fibroblasts to cardiomyocytes. The resultant CiCMs exhibit elevated cardiac marker expression, sarcomeric organization, and improved electrophysiological features and drug responses. We investigated the therapeutic potential of CiCMs reprogrammed in three-dimensional heart extracellular matrix in a rat model of myocardial infarction. Our platform can facilitate the use of CiCMs for regenerative medicine, disease modeling, and drug screening.

Human in vitro neurocardiac coculture (ivNCC) assay development for evaluating cardiac contractility modulation.

Two of the most prominent organ systems, the nervous and the cardiovascular systems, are intricately connected to maintain homeostasis in mammals. Recent years have shown tremendous efforts toward therapeutic modulation of cardiac contractility and electrophysiology by electrical stimulation. Neuronal innervation and cardiac ganglia regulation are often overlooked when developing in vitro models for cardiac devices, but it is likely that peripheral nervous system plays a role in the clinical effects. We developed an in vitro neurocardiac coculture (ivNCC) model system to study cardiac and neuronal interplay using human induced pluripotent stem cell (hiPSC) technology. We demonstrated significant expression and colocalization of cardiac markers including troponin, α-actinin, and neuronal marker peripherin in neurocardiac coculture. To assess functional coupling between the cardiomyocytes and neurons, we evaluated nicotine-induced β-adrenergic norepinephrine effect and found beat rate was significantly increased in ivNCC as compared to monoculture alone. The developed platform was used as a nonclinical model for the assessment of cardiac medical devices that deliver nonexcitatory electrical pulses to the heart during the absolute refractory period of the cardiac cycle, that is, cardiac contractility modulation (CCM) therapy. Robust coculture response was observed at 14 V/cm (5V, 64 mA), monophasic, 2ms pulse duration for pacing and 20 V/cm (7V, 90 mA) phase amplitude, biphasic, 5.14 ms pulse duration for CCM. We observed that the CCM effect and kinetics were more pronounced in coculture as compared to cardiac monoculture, supporting a hypothesis that some part of CCM mechanism of action can be attributed to peripheral nervous system stimulation. This study provides novel characterization of CCM effects on hiPSC-derived neurocardiac cocultures. This innervated human heart model can be further extended to investigate arrhythmic mechanisms, neurocardiac safety, and toxicity post-chronic exposure to materials, drugs, and medical devices. We present data on acute CCM electrical stimulation effects on a functional and optimized coculture using commercially available hiPSC-derived cardiomyocytes and neurons. Moreover, this study provides an in vitro human heart model to evaluate neuronal innervation and cardiac ganglia regulation of contractility by applying CCM pulse parameters that closely resemble clinical setting. This ivNCC platform provides a potential tool for investigating aspects of cardiac and neurological device safety and performance.

Visfatin Amplifies Cardiac Inflammation and Aggravates Cardiac Injury via the NF-κB p65 Signaling Pathway in LPS-Treated Mice.

Background Visfatin is an adipocytokine that has been demonstrated to be involved in cardiovascular diseases. This study aims at determining the role of visfatin in sepsis-induced cardiac injury and identify its possible mechanisms. Methods Dynamic changes in visfatin expression in mice with lipopolysaccharide- (LPS-) induced septicemia were measured. Additionally, mice were pretreated with visfatin and further administered LPS to observe the effects of visfatin on cardiac injury. Finally, septic mice were also pretreated with JSH-23 to investigate whether visfatin regulates cardiac injury via the NF-κB p65 pathway. Results Visfatin expression levels in both the heart and serum were increased in LPS-treated mice and peaked at 6 hours, and visfatin was derived from cardiac macrophages. In septic mice, pretreatment with visfatin reduced the survival rate, worsened cardiac dysfunction, and increased the expression of cardiac injury markers, including creatine kinase myocardial bound (CK-MB) and lactate dehydrogenase (LDH). Treatment with visfatin also increased the infiltration of CD3+ cells and F4/80+ cells, amplified the cardiac inflammatory response, and elevated myocardial cell apoptosis. Treatment with JSH-23 reversed the effects of visfatin in septic mice. Conclusions This study showed that visfatin amplifies the cardiac inflammatory response and aggravates cardiac injury through the p65 signaling pathway. Visfatin may be a clinical target for preventing cardiac injury in sepsis.

Overexpression of PYGO1 promotes early cardiac lineage development in human umbilical cord mesenchymal stromal/stem cells by activating the Wnt/β-catenin pathway.

Cardiovascular disease still has the highest mortality. Gene-modified mesenchymal stromal/stem cells could be a promising therapy. Pygo plays an important role in embryonic development and regulates life activities with a variety of regulatory mechanisms. Therefore, this study aimed to investigate whether the overexpression of the PYGO1 gene can promote the differentiation of human umbilical cord-derived mesenchymal stromal/stem cells (HUC-MSCs) into early cardiac lineage cells and to preliminary explore the relevant mechanisms. In this study, HUC-MSCs were isolated by the explant method and were identified by flow cytometry and differentiation assay, followed by transfected with lentivirus carrying the PYGO1 plasmid. In PYGO1 group (cells were incubated with lentiviral-PYGO1), the mRNA expressions of cardiac differentiation-specific markers (MESP1, NKX2.5, GATA4, MEF2C, ISL1, TBX5, TNNT2, ACTC1, and MYH6 genes) and the protein expressions of NKX2.5 and cTnT were significantly up-regulated compared with the NC group (cells were incubated with lentiviral-empty vector). In addition, the proportion of NKX2.5, GATA4, and cTnT immunofluorescence-positive cells increased with the inducement time. Overexpression of PYGO1 statistically significantly increased the relative luciferase expression level of Topflash plasmid, the protein expression level of β-catenin and the mRNA expression level of CYCLIND1. Compared with the control group, decreased protein levels of NKX2.5 and cTnT were detected in PYGO1 group after application of XAV-939, the specific inhibitor of the canonical Wnt/β-catenin pathway. Our study suggests that overexpression of PYGO1 significantly promotes the differentiation of HUC-MSCs into early cardiac lineage cells, which is regulated by the canonical Wnt/β-catenin signaling.

Timely delivery of cardiac mmRNAs in microfluidics enhances cardiogenic programming of human pluripotent stem cells.

In the last two decades lab-on-chip models, specifically heart-on-chip, have been developed as promising technologies for recapitulating physiological environments suitable for studies of drug and environmental effects on either human physiological or patho-physiological conditions. Most human heart-on-chip systems are based on integration and adaptation of terminally differentiated cells within microfluidic context. This process requires prolonged procedures, multiple steps, and is associated with an intrinsic variability of cardiac differentiation. In this view, we developed a method for cardiac differentiation-on-a-chip based on combining the stage-specific regulation of Wnt/β-catenin signaling with the forced expression of transcription factors (TFs) that timely recapitulate hallmarks of the cardiac development. We performed the overall cardiac differentiation from human pluripotent stem cells (hPSCs) to cardiomyocytes (CMs) within a microfluidic environment. Sequential forced expression of cardiac TFs was achieved by a sequential mmRNAs delivery of first MESP1, GATA4 followed by GATA4, NKX2.5, MEF2C, TBX3, and TBX5. We showed that this optimized protocol led to a robust and reproducible approach to obtain a cost-effective hiPSC-derived heart-on-chip. The results showed higher distribution of cTNT positive CMs along the channel and a higher expression of functional cardiac markers (TNNT2 and MYH7). The combination of stage-specific regulation of Wnt/β-catenin signaling with mmRNAs encoding cardiac transcription factors will be suitable to obtain heart-on-chip model in a cost-effective manner, enabling to perform combinatorial, multiparametric, parallelized and high-throughput experiments on functional cardiomyocytes.

Sodium Alginate/Chitosan Scaffolds for Cardiac Tissue Engineering: The Influence of Its Three-Dimensional Material Preparation and the Use of Gold Nanoparticles.

Natural biopolymer scaffolds and conductive nanomaterials have been widely used in cardiac tissue engineering; however, there are still challenges in the scaffold fabrication, which include enhancing nutrient delivery, biocompatibility and properties that favor the growth, maturation and functionality of the generated tissue for therapeutic application. In the present work, different scaffolds prepared with sodium alginate and chitosan (alginate/chitosan) were fabricated with and without the addition of metal nanoparticles and how their fabrication affects cardiomyocyte growth was evaluated. The scaffolds (hydrogels) were dried by freeze drying using calcium gluconate as a crosslinking agent, and two types of metal nanoparticles were incorporated, gold (AuNp) and gold plus sodium alginate (AuNp+Alg). A physicochemical characterization of the scaffolds was carried out by swelling, degradation, permeability and infrared spectroscopy studies. The results show that the scaffolds obtained were highly porous (>90%) and hydrophilic, with swelling percentages of around 3000% and permeability of the order of 1 × 10−8 m2. In addition, the scaffolds proposed favored adhesion and spheroid formation, with cardiac markers expression such as tropomyosin, troponin I and cardiac myosin. The incorporation of AuNp+Alg increased cardiac protein expression and cell proliferation, thus demonstrating their potential use in cardiac tissue engineering.

Abstract P1138: Combination Of Pluripotency And Cardiac Transcription Factors Reprogram Skin Fibroblasts Into Cardiomyocytes

Direct reprogramming of autologous somatic cells into cardiomyocytes is a novel approach which is being employed for cardiac regeneration. Multiple studies have combined different cardiac-specific factors that could directly reprogram cardiac fibroblasts into induced cardiomyocyte-like cells. However, this approach needs heart biopsy for the isolation of autologous fibroblasts which involves extensive surgical procedures that require precision. In this study, we aimed to develop an approach which can directly reprogram somatic cells into cardiomyocyte-like cells using a combination of pluripotency and cardiac transcription factors. Skin fibroblasts were isolated from rat neonatal pups and were induced into cardiomyocyte-like cells using non-viral integration of cardiac transcription factors (GATA4, Mef2c and Nkx2.5) and OKSIM plasmid carrying the iPSC factors, Sox2, Oct4, Klf4 and cMyc. After 72 h, cells were analyzed for the expression of cardiac markers by qPCR and immunocytochemistry. Gene expression analysis showed significantly higher expression of cardiac markers GATA4, cMHC, Mef2c, cTnT, cTnI, and Nkx2.5 Immunostaining confirmed the expression of cardiac proteins GATA4, cMHC, cTnT, cTnI, and Nkx2.5. These results imply that the transfected cells started differentiating towards cardiac lineage. Transfected cells were also transplanted in rat myocardial infarction (MI) model immediately after ligation of left ventricle descending (LAD) artery. After 2 and 4 weeks of cell transplantation, the animals were assessed for cardiac function via echocardiography. They showed significant improvement in the cardiac function as compared to MI and non-transfected groups. After 4 weeks, the hearts were harvested, and histological analysis was performed for the assessment of fibrosis and left ventricular wall thickness. Rats transplanted with transfected cells showed significantly reduced fibrosis and increased wall thickness as compared to MI and non-transfected groups. In conclusion, transient and combined expression of cardiac transcription and iPSC factors in neonatal somatic cells leads to the transdifferentiation of skin cells into myogenic lineage. This approach can be useful for future therapeutic application for cardiovascular diseases.

Vaspin Alleviates Sepsis-Induced Cardiac Injury and Cardiac Inflammation by Inhibiting Kallikrein 7 in Mice.

Background Vaspin is an important adipokine that is involved in cardiovascular diseases. This study is aimed at investigating whether vaspin participates in sepsis-induced cardiac injury and explored the possible mechanism. Methods First, cecal ligation and puncture (CLP) and lipopolysaccharide (LPS) were used to establish a mouse model of sepsis, and cardiac vaspin expression was examined. In addition, after pretreatment with vaspin or phosphate-buffered saline (PBS), wild-type (WT) mice underwent CLP to establish a septic model and received sham as a control. Finally, WT mice and kallikrein 7 (KLK7-/-) mice were underwent CLP with or without vaspin pretreatment. Results Mice that underwent CLP and were administered LPS exhibited increased vaspin expression in both the heart and serum compared with sham- or saline-treated mice. In CLP mice, pretreatment with vaspin reduced mortality and alleviated the expression of cardiac injury markers and cardiac dysfunction. In addition, vaspin reduced the cardiac levels of CD45+ cells and CD68+ cells, alleviated the cardiac inflammatory response, and reduced cardiomyocyte apoptosis. The protective effects of vaspin on CLP mice were masked by the deletion of KLK7, which was demonstrated to be a downstream signal of vaspin. Conclusions Vaspin alleviates cardiac inflammation and plays a protective role in sepsis-induced cardiac injury by reducing KLK7 expression.

Transcriptomic analysis and comparative characterization of rat H9C2, human AC16 and murine HL-1 cardiac cell lines

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Union's Horizon 2020 research and innovation programme National Research, Development and Innovation Fund of Hungary Background Cardiac cell lines and primary cell cultures are widely used to model various cardiovascular diseases in vitro. Despite the increasing number of publications using these models, limitations of these cell lines are still undetermined. Purpose The aim of our study was to compare the most commonly used cardiac cell lines to primary cultures and to mature cardiac tissues by transcriptomic analysis and morphological characterization. Methods H9C2 (rat), AC16 (human) and HL-1 (mouse) cardiac cell lines were differentiated towards a phenotype more resembling cardiomyocytes, by methods most widely used in the literature, and cells were harvested at stages of proliferation and differentiation. Whole left ventricular tissue, neonatal primary cardiac myocytes isolated from mice and rats, or human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) were applied as references. Transcriptome analysis and immunocytochemical detection of cardiac structural proteins were performed on all cell models. Results RNA expression of cardiac markers (e.g. Tnnt2, Ryr2, Tnni3) was markedly lower in cell lines compared to primary cells or hiPSC-CM and adult tissue controls. Differentiation procedures induced a significant increase in cardiac- and decrease in embryonic markers in AC16 and H9C2 lines; however, the overall expression pattern of investigated genes in all cell lines showed significant differences in comparison to corresponding myocardium or primary cultures. Immunocytochemistry confirmed low expressions of structural protein alpha-actinin and troponin I in cell lines. Conclusion Expression patterns of cardiomyocyte markers and mRNA profile indicates low-to-moderate similarity of cell lines to primary cells/cardiac tissues regardless the differentiation protocol used. These limitations should be taken into account while choosing cells as in vitro platforms to model cardiomyocytes and cardiovascular diseases.

Potential Protective Role of 14-3-3 Protein in Pathological Cardiac Hypertrophy Through The Regulation of Endoplasmic Reticulum Stress: Role of Calreticulin

Introduction: 14-3-3 protein plays an important role in protecting cardiac cells from hypertrophy and endoplasmic reticulum stress during pressure overload elicited by aortic banding (AB) surgery; however, the relation among these protective roles is largely unknown. Methods: We investigated the in vivo role of 14-3-3 protein in two protocols involving C57/BL-6 mice and dominant negative (DN) 14-3-3η mice subjected to three- or seven-days pressure overload stimulation by applying AB surgery. The protein expressions of cardiac hypertrophy and ER stress markers, including atrial natriuretic peptide (ANP), galectin-3, glucose-regulated protein (GRP)78, calreticulin as well as 14-3-3 protein was analyzed by western blot. Results: Three- or seven-day pressure overload stimulation significantly increased the heart weight/body weight (HW/BW) ratio; cardiomyocyte diameter; and the protein expression levels of ANP, GRP78 as well as 14-3-3 in the C57/BL-6 mice. Partial inactivation of 14-3-3 protein in the DN 14-3-3η mice significantly increased the protein expression of ANP, Galectin-3, GRP78, and calreticulin after three- or seven-days AB surgery. Conclusion: These results suggest that 14-3-3 protein, as a molecular chaperone, protects against pathological cardiac hypertrophy, at least in part, by maintaining the normal ER function through the regulation of GRP78 and calreticulin.

LncRNA KCNQ1OT1 attenuates myocardial injury induced by hip fracture via regulating of miR-224-3p/GATA4 axis

ObjectiveThis article aims to discuss the role of l KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1) in myocardial injury caused by a hip fracture and further investigate its potential molecular mechanisms. MethodsX-Ray and H&E staining are used to observe hip fracture and pathological changes of myocardial tissue. ELISA and kits are used to detect inflammatory cytokines, lactate dehydrogenase (LDH), and creatine kinase (CK) in serum. The proliferation and apoptosis of H9c2 are determined by CCK-8 and flow cytometry. RT-qPCR and Western blot are applied to quantitatively assess the expression of related genes. Bioinformatics analysis is performed to search the downstream target of KCNQ1OT1 and miR-224-3p. Furthermore, the interaction is verified by a luciferase reporter assay. ResultsA hip fracture model was successfully established. The high expression of inflammatory cytokines and cardiac injury markers indicated that hip fracture successfully induced myocardial injury. In TNF-ɑ treated cardiomyocyte model, high expression of KCNQ1OT1 promoted H9c2 cell proliferation and inhibited apoptosis. Furthermore, in the myocardial injury model rats induced by hip fracture, a high expression of KCNQ1OT1 reduced pathological damage in the myocardial tissue. Further research illustrated that miR-224-3p was the direct target of KCNQ1OT1, and GATA4 was the direct target of miR-224-3p. Importantly, functional research findings indicated that KCNQ1OT1 regulated myocardial injury caused by hip fracture via targeting the miR-224-3p/GATA4 axis. ConclusionOur study demonstrates that the KCNQ1OT1 suppresses myocardial injury via mediating miR-224-3p/GATA4, which provides a latent target for myocardial injury treatment.

Activation of the endocannabinoid system mediates cardiac hypertrophy induced by rosiglitazone.

Rosiglitazone (RSG) is a synthetic agonist of peroxisome proliferator-activated receptor-γ (PPARγ), which plays a central role in the regulation of metabolism. Meta-analyses have suggested that RSG is associated with increased cardiovascular risk. However, the mechanisms underlying such adverse cardiac effects are still poorly understood. Here, we found that activation of PPARγ by RSG stimulated the endocannabinoid system (ECS), a membrane lipid signaling system, which induced cardiac hypertrophy. In neonatal rat cardiomyocytes, RSG increased the level of anandamide (AEA); upregulated the expression of N-acyl phosphatidylethanolamine phospholipase D (NapePLD), a key enzyme for AEA synthesis; and downregulated the expression of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of AEA. Importantly, PPARγ activation increased the expression of cannabinoid receptor type 1 (CB1) through an identified binding site for PPARγ in the CB1 promoter region. Moreover, both the in vitro and in vivo results showed that inhibition of the ECS by rimonabant, an antagonist of CB1, attenuated RSG-induced cardiac hypertrophy, as indicated by decreased expression of cardiac hypertrophy markers (ANP and BNP), deactivation of the mTOR pathway, and decreased cardiomyocyte size. Thus, these results demonstrated that the ECS functions as a novel target of PPARγ and that the AEA/CB1/mTOR axis mediates RSG-induced cardiac remodeling.

Systematic transcriptomic and phenotypic characterization of human and murine cardiac myocyte cell lines and primary cardiomyocytes reveals serious limitations and low resemblances to adult cardiac phenotype

BackgroundCardiac cell lines and primary cells are widely used in cardiovascular research. Despite increasing number of publications using these models, comparative characterization of these cell lines has not been performed, therefore, their limitations are undetermined. We aimed to compare cardiac cell lines to primary cardiomyocytes and to mature cardiac tissues in a systematic manner. Methods and resultsCardiac cell lines (H9C2, AC16, HL-1) were differentiated with widely used protocols. Left ventricular tissue, neonatal primary cardiomyocytes, and human induced pluripotent stem cell-derived cardiomyocytes served as reference tissue or cells. RNA expression of cardiac markers (e.g. Tnnt2, Ryr2) was markedly lower in cell lines compared to references. Differentiation induced increase in cardiac- and decrease in embryonic markers however, the overall transcriptomic profile and annotation to relevant biological processes showed consistently less pronounced cardiac phenotype in all cell lines in comparison to the corresponding references. Immunocytochemistry confirmed low expressions of structural protein sarcomeric alpha-actinin, troponin I and caveolin-3 in cell lines. Susceptibility of cell lines to sI/R injury in terms of viability as well as mitochondrial polarization differed from the primary cells irrespective of their degree of differentiation. ConclusionExpression patterns of cardiomyocyte markers and whole transcriptomic profile, as well as response to sI/R, and to hypertrophic stimuli indicate low-to-moderate similarity of cell lines to primary cells/cardiac tissues regardless their differentiation. Low resemblance of cell lines to mature adult cardiac tissue limits their potential use. Low translational value should be taken into account while choosing a particular cell line to model cardiomyocytes.