Transcriptome-based prediction of drugs, inhibiting cardiomyogenesis in human induced pluripotent stem cells.

Animal studies for embryotoxicity evaluation of potential therapeutics and environmental factors are complex, costly, and time-consuming. Often, studies are not of human relevance because of species differences. In the present study, we recapitulated the process of cardiomyogenesis in human induced pluripotent stem cells (hiPSCs) by modulation of the Wnt signaling pathway to identify a key cardiomyogenesis gene signature that can be applied to identify compounds and/or stress factors compromising the cardiomyogenesis process. Among the 23 tested teratogens and 16 non-teratogens, we identified three retinoids including 13-cis-retinoic acid that completely block the process of cardiomyogenesis in hiPSCs. Moreover, we have identified an early gene signature consisting of 31 genes and associated biological processes that are severely affected by the retinoids. To predict the inhibitory potential of teratogens and non-teratogens in the process of cardiomyogenesis we established the "Developmental Cardiotoxicity Index" (CDI31g) that accurately differentiates teratogens and non-teratogens to do or do not affect the differentiation of hiPSCs to functional cardiomyocytes.

Microgravity-induced stress mechanisms in human stem cell-derived cardiomyocytes.

Exposure to outer space microgravity poses a risk for the development of various pathologies including cardiovascular disease. To study this, we derived cardiomyocytes (CMs) from human-induced pluripotent stem cells and exposed them to simulated microgravity (SMG). We combined different "omics" and chromosome conformation capture technologies with live-cell imaging of various transgenic lines to discover that SMG impacts on the contractile velocity and function of CMs via the induction of senescence processes. This is linked to SMG-induced changes of reactive oxygen species (ROS) generation and energy metabolism by mitochondria. Taken together, we uncover a microgravity-controlled axis causing contractile dysfunctions to CMs. Our findings can contribute to the design of preventive and therapeutic strategies against senescence-associated disease.

Live-Cell Imaging of the Contractile Velocity and Transient Intracellular Ca2+ Fluctuations in Human Stem Cell-Derived Cardiomyocytes.

Live-cell imaging techniques are essential for acquiring vital physiological and pathophysiological knowledge to understand and treat heart disease. For live-cell imaging of transient alterations of [Ca2+]i in human cardiomyocytes, we engineered human-induced pluripotent stem cells carrying a genetically-encoded Ca2+-indicator (GECI). To monitor sarcomere shortening and relaxation in cardiomyocytes in real-time, we generated a α-cardiac actinin (ACTN2)-copepod (cop) green fluorescent protein (GFP+)-human-induced pluripotent stem cell line by using the CRISPR-Cas9 and a homology directed recombination approach. The engineered human-induced pluripotent stem cells were differentiated in transgenic GECI-enhanced GFP+-cardiomyocytes and ACTN2-copGFP+-cardiomyocytes, allowing real-time imaging of [Ca2+]i transients and live recordings of the sarcomere shortening velocity of ACTN2-copGFP+-cardiomyocytes. We developed a video analysis software tool to quantify various parameters of sarcoplasmic Ca2+ fluctuations recorded during contraction of cardiomyocytes and to calculate the contraction velocity of cardiomyocytes in the presence and absence of different drugs affecting cardiac function. Our cellular and software tool not only proved the positive and negative inotropic and lusitropic effects of the tested cardioactive drugs but also quantified the expected effects precisely. Our platform will offer a human-relevant in vitro alternative for high-throughput drug screenings, as well as a model to explore the underlying mechanisms of cardiac diseases.

Correction: Nemade, H.; et al. Cyclooxygenases Inhibitors Efficiently Induce Cardiomyogenesis in Human Pluripotent Stem Cells. Cells 2020, 9, 554.

The authors wish to make the following corrections to this paper [...].

Cyclooxygenases Inhibitors Efficiently Induce Cardiomyogenesis in Human Pluripotent Stem Cells.

Application of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is limited by the challenges in their efficient differentiation. Recently, the Wingless (Wnt) signaling pathway has emerged as the key regulator of cardiomyogenesis. In this study, we evaluated the effects of cyclooxygenase inhibitors on cardiac differentiation of hPSCs. Cardiac differentiation was performed by adherent monolayer based method using 4 hPSC lines (HES3, H9, IMR90, and ES4SKIN). The efficiency of cardiac differentiation was evaluated by flow cytometry and RT-qPCR. Generated hPSC-CMs were characterised using immunocytochemistry, electrophysiology, electron microscopy, and calcium transient measurements. Our data show that the COX inhibitors Sulindac and Diclofenac in combination with CHIR99021 (GSK-3 inhibitor) efficiently induce cardiac differentiation of hPSCs. In addition, inhibition of COX using siRNAs targeted towards COX-1 and/or COX-2 showed that inhibition of COX-2 alone or COX-1 and COX-2 in combination induce cardiomyogenesis in hPSCs within 12 days. Using IMR90-Wnt reporter line, we showed that inhibition of COX-2 led to downregulation of Wnt signalling activity in hPSCs. In conclusion, this study demonstrates that COX inhibition efficiently induced cardiogenesis via modulation of COX and Wnt pathway and the generated cardiomyocytes express cardiac-specific structural markers as well as exhibit typical calcium transients and action potentials. These cardiomyocytes also responded to cardiotoxicants and can be relevant as an in vitro cardiotoxicity screening model.

Parabolic, Flight-Induced, Acute Hypergravity and Microgravity Effects on the Beating Rate of Human Cardiomyocytes.

Functional studies of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hCMs) under different gravity conditions contribute to aerospace medical research. To study the effects of altered gravity on hCMs, we exposed them to acute hypergravity and microgravity phases in the presence and absence of the ß-adrenoceptor isoprenalin (ISO), L-type Ca2+ channel (LTCC) agonist Bay-K8644, or LTCC blocker nifedipine, and monitored their beating rate (BR). These logistically demanding experiments were executed during the 66th Parabolic Flight Campaign of the European Space Agency. The hCM cultures were exposed to 31 alternating hypergravity, microgravity, and hypergravity phases, each lasting 20-22 s. During the parabolic flight experiment, BR and cell viability were monitored using the xCELLigence real-time cell analyzer Cardio Instrument®. Corresponding experiments were performed on the ground (1 g), using an identical set-up. Our results showed that BR continuously increased during the parabolic flight, reaching a 40% maximal increase after 15 parabolas, compared with the pre-parabolic (1 g) phase. However, in the presence of the LTCC blocker nifedipine, no change in BR was observed, even after 31 parabolas. We surmise that the parabola-mediated increase in BR was induced by the LTCC blocker. Moreover, the increase in BR induced by ISO and Bay-K8644 during the pre-parabola phase was further elevated by 20% after 25 parabolas. This additional effect reflects the positive impact of the parabolas in the absence of both agonists. Our study suggests that acute alterations of gravity significantly increase the BR of hCMs via the LTCC.

Modulation of Differentiation Processes in Murine Embryonic Stem Cells Exposed to Parabolic Flight-Induced Acute Hypergravity and Microgravity.

Embryonic developmental studies under microgravity conditions in space are very limited. To study the effects of short-term altered gravity on embryonic development processes, we exposed mouse embryonic stem cells (mESCs) to phases of hypergravity and microgravity and studied the differentiation potential of the cells using wide-genome microarray analysis. During the 64th European Space Agency's parabolic flight campaign, mESCs were exposed to 31 parabolas. Each parabola comprised phases lasting 22 s of hypergravity, microgravity, and a repeat of hypergravity. On different parabolas, RNA was isolated for microarray analysis. After exposure to 31 parabolas, mESCs (P31 mESCs) were further differentiated under normal gravity (1 g) conditions for 12 days, producing P31 12-day embryoid bodies (EBs). After analysis of the microarrays, the differentially expressed genes were analyzed using different bioinformatic tools to identify developmental and nondevelopmental biological processes affected by conditions on the parabolic flight experiment. Our results demonstrated that several genes belonging to GOs associated with cell cycle and proliferation were downregulated in undifferentiated mESCs exposed to gravity changes. However, several genes belonging to developmental processes, such as vasculature development, kidney development, skin development, and to the TGF-ß signaling pathway, were upregulated. Interestingly, similar enriched and suppressed GOs were obtained in P31 12-day EBs compared with ground control 12-day EBs. Our results show that undifferentiated mESCs exposed to alternate hypergravity and microgravity phases expressed several genes associated with developmental/differentiation and cell cycle processes, suggesting a transition from the undifferentiated pluripotent to a more differentiated stage of mESCs.

Cell death mechanisms of the anti-cancer drug etoposide on human cardiomyocytes isolated from pluripotent stem cells.

Etoposide (ETP) and anthracyclines are applied for wide anti-cancer treatments. However, the ETP-induced cardiotoxicity remains to be a major safety issue and the underlying cardiotoxic mechanisms are not well understood. This study is aiming to unravel the cardiotoxicity profile of ETP in comparison to anthracyclines using physiologically relevant human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). Using xCELLigence real-time cell analyser (RTCA), we found that single high dose of ETP induces irreversible increase in hPSC-CMs beating rate and decrease in beating amplitude. We also identified 58 deregulated genes consisting of 33 upregulated and 25 downregulated genes in hPSC-CMs after ETP treatment. Gene ontology (GO) and pathway analysis showed that most upregulated genes are enriched in GO categories like positive regulation of apoptotic process, regulation of cell death, and mitochondria organization, whereas most downregulated genes were enriched in GO categories like cytoskeletal organization, muscle contraction, and Ca2+ ion homeostasis. Moreover, we also found upregulation in 5 miRNAs (has-miR-486-3p, has-miR-34c-5p, has-miR-4423-3p, has-miR-182-5p, and has-miR-139-5p) which play role in muscle contraction, arginine and proline metabolism, and hypertrophic cardiomyopathy (HCM). Immunostaining and transmission electron microscopy also confirmed the cytoskeletal and mitochondrial damage in hPSC-CMs treated with ETP, as well as noticeable alterations in intracellular calcium handling and mitochondrial membrane potential were also observed. The apoptosis inhibitor, Pifithrin-α, found to protect hPSC-CMs from ETP-induced cardiotoxicity, whereas hPSC-CMs treated with ferroptosis inhibitor, Liproxstatin-1, showed significant recovery in hPSC-CMs functional properties like beating rate and amplitude after ETP treatment. We suggest that the damage to mitochondria is a major contributing factor involved in ETP-induced cardiotoxicity and the activation of the p53-mediated ferroptosis pathway by ETP is likely the critical pathway in ETP-induced cardiotoxicity. We also conclude that the genomic biomarkers identified in this study will significantly contribute to develop and predict potential cardiotoxic effects of novel anti-cancer drugs in vitro.

Simulated Microgravity Modulates Differentiation Processes of Embryonic Stem Cells.

BACKGROUND/AIMS: Embryonic developmental studies under microgravity conditions in space are very limited. To study the effects of altered gravity on the embryonic development processes we established an in vitro methodology allowing differentiation of mouse embryonic stem cells (mESCs) under simulated microgravity within a fast-rotating clinostat (clinorotation) and capture of microarray-based gene signatures. METHODS: The differentiating mESCs were cultured in a 2D pipette clinostat. The microarray and bioinformatics tools were used to capture genes that are deregulated by simulated microgravity and their impact on developmental biological processes. RESULTS: The data analysis demonstrated that differentiation of mESCs in pipettes for 3 days resultet to early germ layer differentiation and then to the different somatic cell types after further 7 days of differentiation in the Petri dishes. Clinorotation influences differentiation as well as non-differentiation related biological processes like cytoskeleton related 19 genes were modulated. Notably, simulated microgravity deregulated genes Cyr61, Thbs1, Parva, Dhrs3, Jun, Tpm1, Fzd2 and Dll1 are involved in heart morphogenesis as an acute response on day 3. If the stem cells were further cultivated under normal gravity conditions (1 g) after clinorotation, the expression of cardiomyocytes specific genes such as Tnnt2, Rbp4, Tnni1, Csrp3, Nppb and Mybpc3 on day 10 was inhibited. This correlated well with a decreasing beating activity of the 10-days old embryoid bodies (EBs). Finally, we captured Gadd45g, Jun, Thbs1, Cyr61and Dll1 genes whose expressions were modulated by simulated microgravity and by real microgravity in various reported studies. Simulated microgravity also deregulated genes belonging to the MAP kinase and focal dhesion signal transduction pathways. CONCLUSION: One of the most prominent biological processes affected by simulated microgravity was the process of cardiomyogenesis. The most significant simulated microgravity-affected genes, signal transduction pathways, and biological processes which are relevant for mESCs differentiation have been identified and discussed below.

Effects of combination of aliskiren and pentoxyfylline on renal function in the rat remnant kidney model of chronic renal failure.

OBJECTIVES: The aim was to investigate the nephroprotective effect of combination of aliskiren (ASK), a direct renin inhibitor and pentoxifylline (PTX), inhibitor of tumor necrotic factor-alpha (TNF-alpha), in rat remnant kidney model of chronic kidney disease (CKD). MATERIALS AND METHODS: Nephrectomized (NPX) rats were treated with ASK (10 mg/kg, p.o.), PTX (100 mg/kg, p.o.), and combination of PTX + ASK once daily for 28 days. We have performed analysis of various renal injury parameters after 4 weeks of treatment. RESULTS: Treatment with PTX, ASK and combination showed significant improvement in urea, creatinine and total protein in plasma when compared with vehicle treated group in NPX rats. ASK and combination of PTX + ASK elicited significant reduction in blood pressure but PTX alone did not produce blood pressure reduction. ASK treatment showed significant elevation in TNF-alpha, whereas PTX and ASK + PTX showed significant reduction in TNF-alpha in plasma. Histopathologically, the extent of the kidney injury was similar in NPX + vehicle and NPX + ASK-treated rats. PTX and ASK + PTX-treated group showed lesser extent of kidney injury. There was good correlation of mRNA expression levels of kidney injury molecule-1 and bradykinin B1 receptor data with histopathological findings in kidney samples and elevated TNF-alpha levels in plasma. CONCLUSIONS: We conclude that combination of PTX + ASK may be better therapeutic intervention for nephroprotection in CKD patients.

Blockade of tumor necrosis factor-α converting enzyme (TACE) enhances IL-1ß and IFN-γ via caspase-1 activation: a probable cause for loss of efficacy of TACE inhibitors in humans?

TNF-α converting enzyme (TACE) is a member of the ADAM (a disintegrin and metalloproteinase) family and is known as ADAM17, which processes precursor TNF-α in order to release soluble TNF-α (sTNF-α). Inhibition of TACE has been effective as a strategy to inhibit arthritis in animal models; however, it has not been translated in the clinic due to lack of efficacy or toxicity. We hypothesized that inhibition of TACE may activate a different pro-inflammatory pathway in human. To investigate this, we studied the effect of TACE inhibitor DPC-333 on cytokine levels in concanavalin A (Con A) activated human peripheral blood mononuclear cells (hPBMC). We have also studied the effects of DPC-333 on Con A induced cytokine levels in mice in vivo or in vitro in whole blood assay. DPC-333 treatment significantly up-regulated IL-1ß and IFN-γ in Con A activated hPBMC. In contrast, pre-treatment with DPC-333 effectively suppressed IL-1ß and IFN-γ in mice in vivo or in vitro. Interestingly, DPC-333 was found to up-regulate mRNA expression of caspase-1 in hPBMC in a dose dependent fashion and selective caspase-1 inhibitor completely restored DPC-333 induced IL-1ß and IFN-γ. Furthermore, selective IL-1ß receptor antagonist (anakinra) prevented DPC-333 induced IFN-γ. In conclusion, our data demonstrates that blockade of TACE enhances IL-1ß in a caspase-1 dependent manner in vitro in hPBMC and the elevation of IFN-γ is secondarily mediated via IL-1ß. This novel finding might explain the possible cause behind the loss of efficacy of TACE inhibitors in human.

Retinol-binding protein 4 : a possible role in cardiovascular complications.

BACKGROUND AND PURPOSE: Retinol-binding protein 4 (RBP4) is an adipocyte-secreted hormone proposed to link obesity with insulin resistance. However, the role of RBP4 in cardiovascular complications is yet to be fully understood. The present study is aimed to decipher the association between RBP4 with pro-inflammatory cytokines and low-density lipoprotein (LDL) cholesterol in diet-induced obese and hyperlipidaemic mice. To understand the correlation, rimonabant, an anti-obesity drug, has been used to relieve the atherosclerotic predisposition. EXPERIMENTAL APPROACH: Adipose and/or aortic tissue expressions of RBP4, pro-inflammatory cytokine genes and circulating LDL levels were measured in high fat (HF)-fed female C57BL/6 and high cholesterol (HC)-fed apolipoprotein E3 (ApoE3) Leiden mice. KEY RESULTS: Mice fed a HF diet had a significantly increased adipose expression of RBP4, TNF-α and monocyte chemoattractant protein 1 (MCP-1) and down-regulated adiponectin mRNA levels. A significant increase in aortic RBP4 and MCP-1 expression and circulating levels of LDL and C-reactive protein (CRP) was found in the ApoE3 mice fed a HC diet. Interestingly, rimonabant treatment lowered the elevated aortic RBP4, MCP-1 expressions and significantly reduced the serum levels of LDL, CRP, RBP4 and MCP-1. CONCLUSION AND IMPLICATIONS: Our results indicate that RBP4 is positively associated with markers of inflammation in obese and pro-atherogenic conditions and could play a role in a predisposition to atherosclerosis. Furthermore, our results indicate that rimonabant may improve vascular function by modulating RBP4 along with pro-inflammatory cytokines.

Beneficial effects of carbon monoxide-releasing molecule-2 (CORM-2) on acute doxorubicin cardiotoxicity in mice: role of oxidative stress and apoptosis.

Doxorubicin (DXR) has been used in variety of human malignancies for decades. Despite its efficacy in cancer, clinical usage is limited because of its cardiotoxicity, which has been associated with oxidative stress and apoptosis. Carbon monoxide-releasing molecules (CORMs) have been shown to reduce the oxidative damage and apoptosis. The present study investigated the effects of CORM-2, a fast CO-releaser, against DXR-induced cardiotoxicity in mice using biochemical, histopathological and gene expression approaches. CORM-2 (3, 10 and 30 mg/kg/day) was administered intraperitoneally (i.p.) for 10 days and terminated the study on day 11. DXR (20 mg/kg, i.p.) was injected before 72 h of termination. Mice treated with DXR showed cardiotoxicity as evidenced by elevation of serum creatine kinase (CK) and lactate dehydrogenase (LDH), tissue malondialdehyde (MDA), caspase-3 and decrease the level of total antioxidant status (TAS) in heart tissues. Pre- and post-treatment with CORM-2 (30 mg/kg, i.p.) elicited significant improvement in CK, LDH, MDA, caspase-3 and TAS levels. Histopathological studies showed that cardiac damage with DXR has been reversed with CORM-2+DXR treatment. There was dramatic decrease in hematological count in DXR-treated mice, which has been improved with CORM-2. Furthermore, there was also elevation of mRNA expression of heme oxygenase-1, hypoxia inducible factor-1 alpha, vascular endothelial growth factor and decrease in inducible-nitric oxide synthase expression upon treatment with CORM-2 that might be linked to cardioprotection. These data suggest that CORM-2 treatment provides cardioprotection against acute doxorubicin-induced cardiotoxicity in mice and this effect may be attributed to CORM-2-mediated antioxidant and anti-apoptotic properties.