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.

BACKGROUND: Cardiac 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 RESULTS: Cardiac 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. CONCLUSION: Expression 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.

Helium Conditioning Increases Cardiac Fibroblast Migration Which Effect Is Not Propagated via Soluble Factors or Extracellular Vesicles.

Helium inhalation induces cardioprotection against ischemia/reperfusion injury, the cellular mechanism of which remains not fully elucidated. Extracellular vesicles (EVs) are cell-derived, nano-sized membrane vesicles which play a role in cardioprotective mechanisms, but their function in helium conditioning (HeC) has not been studied so far. We hypothesized that HeC induces fibroblast-mediated cardioprotection via EVs. We isolated neonatal rat cardiac fibroblasts (NRCFs) and exposed them to glucose deprivation and HeC rendered by four cycles of 95% helium + 5% CO2 for 1 h, followed by 1 h under normoxic condition. After 40 h of HeC, NRCF activation was analyzed with a Western blot (WB) and migration assay. From the cell supernatant, medium extracellular vesicles (mEVs) were isolated with differential centrifugation and analyzed with WB and nanoparticle tracking analysis. The supernatant from HeC-treated NRCFs was transferred to naïve NRCFs or immortalized human umbilical vein endothelial cells (HUVEC-TERT2), and a migration and angiogenesis assay was performed. We found that HeC accelerated the migration of NRCFs and did not increase the expression of fibroblast activation markers. HeC tended to decrease mEV secretion of NRCFs, but the supernatant of HeC or the control NRCFs did not accelerate the migration of naïve NRCFs or affect the angiogenic potential of HUVEC-TERT2. In conclusion, HeC may contribute to cardioprotection by increasing fibroblast migration but not by releasing protective mEVs or soluble factors from cardiac fibroblasts.

Calcium Ionophore-Induced Extracellular Vesicles Mediate Cytoprotection against Simulated Ischemia/Reperfusion Injury in Cardiomyocyte-Derived Cell Lines by Inducing Heme Oxygenase 1.

Cardioprotection against ischemia/reperfusion injury is still an unmet clinical need. The transient activation of Toll-like receptors (TLRs) has been implicated in cardioprotection, which may be achieved by treatment with blood-derived extracellular vesicles (EVs). However, since the isolation of EVs from blood takes considerable effort, the aim of our study was to establish a cellular model from which cardioprotective EVs can be isolated in a well-reproducible manner. EV release was induced in HEK293 cells with calcium ionophore A23187. EVs were characterized and cytoprotection was assessed in H9c2 and AC16 cell lines. Cardioprotection afforded by EVs and its mechanism were investigated after 16 h simulated ischemia and 2 h reperfusion. The induction of HEK293 cells by calcium ionophore resulted in the release of heterogenous populations of EVs. In H9c2 and AC16 cells, stressEVs induced the downstream signaling of TLR4 and heme oxygenase 1 (HO-1) expression in H9c2 cells. StressEVs decreased necrosis due to simulated ischemia/reperfusion injury in H9c2 and AC16 cells, which was independent of TLR4 induction, but not that of HO-1. Calcium ionophore-induced EVs exert cytoprotection by inducing HO-1 in a TLR4-independent manner.

Effect of hypercholesterolemia on circulating and cardiomyocyte-derived extracellular vesicles.

Hypercholesterolemia (HC) induces, propagates and exacerbates cardiovascular diseases via various mechanisms that are yet not properly understood. Extracellular vesicles (EVs) are involved in the pathomechanism of these diseases. To understand how circulating or cardiac-derived EVs could affect myocardial functions, we analyzed the metabolomic profile of circulating EVs, and we performed an in-depth analysis of cardiomyocyte (CM)-derived EVs in HC. Circulating EVs were isolated with Vezics technology from male Wistar rats fed with high-cholesterol or control chow. AC16 human CMs were treated with Remembrane HC supplement and EVs were isolated from cell culture supernatant. The biophysical properties and the protein composition of CM EVs were analyzed. THP1-ASC-GFP cells were treated with CM EVs, and monocyte activation was measured. HC diet reduced the amount of certain phosphatidylcholines in circulating EVs, independently of their plasma level. HC treatment significantly increased EV secretion of CMs and greatly modified CM EV proteome, enriching several proteins involved in tissue remodeling. Regardless of the treatment, CM EVs did not induce the activation of THP1 monocytes. In conclusion, HC strongly affects the metabolome of circulating EVs and dysregulates CM EVs, which might contribute to HC-induced cardiac derangements.

Ergolide mediates anti-cancer effects on metastatic uveal melanoma cells and modulates their cellular and extracellular vesicle proteomes.

Background: Uveal melanoma is a poor prognosis cancer. Ergolide, a sesquiterpene lactone isolated from Inula Brittanica, exerts anti-cancer properties. The objective of this study was to 1) evaluate whether ergolide reduced metastatic uveal melanoma (MUM) cell survival/viability in vitro and in vivo; and 2) to understand the molecular mechanism of ergolide action. Methods: Ergolide bioactivity was screened via long-term proliferation assay in UM/MUM cells and in zebrafish MUM xenograft models. Mass spectrometry profiled proteins modulated by ergolide within whole cell or extracellular vesicle (EVs) lysates of the OMM2.5 MUM cell line. Protein expression was analyzed by immunoblots and correlation analyses to UM patient survival used The Cancer Genome Atlas (TCGA) data. Results: Ergolide treatment resulted in significant, dose-dependent reductions (48.5 to 99.9%; p<0.0001) in OMM2.5 cell survival in vitro and of normalized primary zebrafish xenograft fluorescence (56%; p<0.0001) in vivo, compared to vehicle controls. Proteome-profiling of ergolide-treated OMM2.5 cells, identified 5023 proteins, with 52 and 55 proteins significantly altered at 4 and 24 hours, respectively ( p<0.05; fold-change >1.2). Immunoblotting of heme oxygenase 1 (HMOX1) and growth/differentiation factor 15 (GDF15) corroborated the proteomic data. Additional proteomics of EVs isolated from OMM2.5 cells treated with ergolide, detected 2931 proteins. There was a large overlap with EV proteins annotated within the Vesiclepedia compendium. Within the differentially expressed proteins, the proteasomal pathway was primarily altered. Interestingly, BRCA2 and CDKN1A Interacting Protein (BCCIP) and Chitinase Domain Containing 1 (CHID1), were the only proteins significantly differentially expressed by ergolide in both the OMM2.5 cellular and EV isolates and they displayed inverse differential expression in the cells versus the EVs. Conclusions: Ergolide is a novel, promising anti-proliferative agent for UM/MUM. Proteomic profiling of OMM2.5 cellular/EV lysates identified candidate pathways elucidating the action of ergolide and putative biomarkers of UM, that require further examination.

The most common form of adult eye cancer is uveal melanoma (UM). Once UM cancer cells spread to organs in the rest of the body, metastatic UM (MUM), there is a poor prognosis for patients with only one approved drug treatment. Hence, it is vital to better understand the cellular and extracellular proteins that regulate UM pathology in order to uncover biomarkers of disease and therapeutic targets. In this original study, we demonstrate a compound called ergolide is capable of severely reducing the metabolic activity and growth of UM cancer cells, grown as isolated monolayers. Ergolide was also able to reduce the growth of human MUM cells growing as tumors in transplanted zebrafish larvae. We identify that ergolide alters specific proteins found in the human UM cells. These proteins once analyzed in detail offer opportunities to understand how new treatment strategies can be developed for UM.

COP1 contributes to UVB-induced signaling in human keratinocytes.

UVB irradiation has been shown to trigger a broad range of changes in gene expression in human skin; however, factors governing these events are still not well understood. In this study, we show that human constitutive photomorphogenic protein-1 (huCOP1), an E3 ligase, contributes to the orchestration of UVB response of keratinocytes. Accordingly, our data show that (i) huCOP1 protein is expressed both in the nucleus and in the cytoplasm of cultured keratinocytes, (ii) UVB reduces the levels of the huCOP1 mRNA and protein, and (iii) induces changes in the subcellular localization of huCOP1. Finally, we show that gene-specific silencing of huCOP1 induces the accumulation of the tumor suppressor p53 protein, which is further increased after UVB irradiation.