Meta-analysis of whole-genome gene expression datasets assessing the effects of IDH1 and IDH2 mutations in isogenic disease models

Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are oncogenic drivers to a variable extent in several tumors, including gliomas, acute myeloid leukemia (AML), cholangiocarcinoma, melanoma, and thyroid carcinoma. The pathobiological effects of these mutations vary considerably, impeding the identification of common expression profiles. We performed an expression meta-analysis between IDH-mutant (IDHmut) and IDH-wild-type (IDHwt) conditions in six human and mouse isogenic disease models. The datasets included colon cancer cells, glioma cells, heart tissue, hepatoblasts, and neural stem cells. Among differentially expressed genes (DEGs), serine protease 23 (PRSS23) was upregulated in four datasets, i.e., in human colon carcinoma cells, mouse heart tissue, mouse neural stem cells, and human glioma cells. Carbonic anhydrase 2 (CA2) and prolyl 3-hydroxylase 2 (P3H2) were upregulated in three datasets, and SOX2 overlapping transcript (SOX2-OT) was downregulated in three datasets. The most significantly overrepresented protein class was termed intercellular signal molecules. An additional DEG set contained genes that were both up- and downregulated in different datasets and included oxidases and extracellular matrix structural proteins as the most significantly overrepresented protein classes. In conclusion, this meta-analysis provides a comprehensive overview of the expression effects of IDH mutations shared between different isogenic disease models. The generated dataset includes biomarkers, e.g., PRSS23 that may gain relevance for further research or clinical applications in IDHmut tumors.

Decrease of Pdzrn3 is required for heart maturation and protects against heart failure

Heart failure is the final common stage of most cardiopathies. Cardiomyocytes (CM) connect with others via their extremities by intercalated disk protein complexes. This planar and directional organization of myocytes is crucial for mechanical coupling and anisotropic conduction of the electric signal in the heart. One of the hallmarks of heart failure is alterations in the contact sites between CM. Yet no factor on its own is known to coordinate CM polarized organization. We have previously shown that PDZRN3, an ubiquitine ligase E3 expressed in various tissues including the heart, mediates a branch of the Planar cell polarity (PCP) signaling involved in tissue patterning, instructing cell polarity and cell polar organization within a tissue. PDZRN3 is expressed in the embryonic mouse heart then its expression dropped significantly postnatally corresponding with heart maturation and CM polarized elongation. A moderate CM overexpression of Pdzrn3 (Pdzrn3 OE) during the first week of life, induced a severe eccentric hypertrophic phenotype with heart failure. In models of pressure-overload stress heart failure, CM-specific Pdzrn3 knockout showed complete protection against degradation of heart function. We reported that Pdzrn3 signaling induced PKC ζ expression, c-Jun nuclear translocation and a reduced nuclear ß catenin level, consistent markers of the planar non-canonical Wnt signaling in CM. We then show that subcellular localization (intercalated disk) of junction proteins as Cx43, ZO1 and Desmoglein 2 was altered in Pdzrn3 OE mice, which provides a molecular explanation for impaired CM polarization in these mice. Our results reveal a novel signaling pathway that controls a genetic program essential for heart maturation and maintenance of overall geometry, as well as the contractile function of CM, and implicates PDZRN3 as a potential therapeutic target for the prevention of human heart failure.

Thrombospondin-1 expression and modulation of Wnt and hippo signaling pathways during the early phase of Trypanosoma cruzi infection of heart endothelial cells

The protozoan parasite, Trypanosoma cruzi, causes severe morbidity and mortality in afflicted individuals. Approximately 30% of T. cruzi infected individuals present with cardiac pathology. The invasive forms of the parasite are carried in the vascular system to infect other cells of the body. During transportation, the molecular mechanisms by which the parasite signals and interact with host endothelial cells (EC) especially heart endothelium is currently unknown. The parasite increases host thrombospondin-1 (TSP1) expression and activates the Wnt/β-catenin and hippo signaling pathways during the early phase of infection. The links between TSP1 and activation of the signaling pathways and their impact on parasite infectivity during the early phase of infection remain unknown. To elucidate the significance of TSP1 function in YAP/β-catenin colocalization and how they impact parasite infectivity during the early phase of infection, we challenged mouse heart endothelial cells (MHEC) from wild type (WT) and TSP1 knockout mice with T. cruzi and evaluated Wnt signaling, YAP/β-catenin crosstalk, and how they affect parasite infection. We found that in the absence of TSP1, the parasite induced the expression of Wnt-5a to a maximum at 2 h (1.73±0.13), P< 0.001 and enhanced the level of phosphorylated glycogen synthase kinase 3β at the same time point (2.99±0.24), P<0.001. In WT MHEC, the levels of Wnt-5a were toned down and the level of p-GSK-3β was lowest at 2 h (0.47±0.06), P< 0.01 compared to uninfected control. This was accompanied by a continuous significant increase in the nuclear colocalization of β-catenin/YAP in TSP1 KO MHEC with a maximum Pearson correlation coefficient of (0.67±0.02), P< 0.05 at 6 h. In WT MHEC, the nuclear colocalization of β-catenin/YAP remained steady and showed a reduction at 6 h (0.29±0.007), P< 0.05. These results indicate that TSP1 plays an important role in regulating β-catenin/YAP colocalization during the early phase of T. cruzi infection. Importantly, dysregulation of this crosstalk by pre-incubation of WT MHEC with a β-catenin inhibitor, endo-IWR 1, dramatically reduced the level of infection of WT MHEC. Parasite infectivity of inhibitor treated WT MHEC was similar to the level of infection of TSP1 KO MHEC. These results indicate that the β-catenin pathway induced by the parasite and regulated by TSP1 during the early phase of T. cruzi infection is an important potential therapeutic target, which can be explored for the prophylactic prevention of T. cruzi infection.

The Insulin-Like Growth Factor Signalling Pathway in Cardiac Development and Regeneration

Insulin and Insulin-like growth factors (IGFs) perform key roles during embryonic development, regulating processes of cell proliferation and survival. The IGF signalling pathway comprises two IGFs (IGF1, IGF2), two IGF receptors (IGFR1, IGFR2), and six IGF binding proteins (IGFBPs) that regulate IGF transport and availability. The IGF signalling pathway is essential for cardiac development. IGF2 is the primary mitogen inducing ventricular cardiomyocyte proliferation and morphogenesis of the compact myocardial wall. Conditional deletion of the Igf1r and the insulin receptor (Insr) genes in the myocardium results in decreased cardiomyocyte proliferation and ventricular wall hypoplasia. The significance of the IGF signalling pathway during embryonic development has led to consider it as a candidate for adult cardiac repair and regeneration. In fact, paracrine IGF2 plays a key role in the transient regenerative ability of the newborn mouse heart. We aimed to review the current knowledge about the role played by the IGF signalling pathway during cardiac development and also the clinical potential of recapitulating this developmental axis in regeneration of the adult heart.

Influence of Low Concentrations of Carbon Monoxide on Metabolism of Isolated Heart under Conditions of Ischemia-Reperfusion

The purpose of the study was to determine the effect of different concentrations of carbon monoxide on the metabolism of isolated mice hearts. Materials and methods. To elucidate the effect of low concentrations of carbon monoxide on the myocardium, we performed retrograde perfusion of isolated hearts of laboratory mice with Krebs-Henseleit solution, which was saturated with carbon monoxide for 5, 10, and 30 minutes. We then determined how different concentrations of carbon monoxide affected coronary volumetric flow rate, myocardial glucose and calcium uptake, creatinine release, and aspartate aminotransferase release. During perfusion, R-wave amplitude and R-R interval were measured using an electrocardiograph. To determine the effect of ischemia on the heart muscle during perfusion with solutions of different concentrations, we measured the area of the affected myocardium after staining with 2,3,5-triphenyltetrazolium chloride. Results and discussion. After these studies, it was found that different concentrations of carbon monoxide had a dose-dependent effect on the isolated mouse heart. However, the dependence of the effects does not follow the pattern «lowest concentration – lowest effect». At the same time, an increase in concentration did not mean an increase in adverse effects on the myocardium. Even on the contrary, the smallest concentration led to increased signs of ischemic myocardial damage. In particular, the use of the solution, through which carbon monoxide was passed for 5 minutes, caused vasoconstrictor effect during perfusion. At the end of reperfusion, vasoconstrictor effect was observed after using a solution through which carbon monoxide was passed for 10 minutes. Increased glucose uptake was observed in the group with 30-minute carbon monoxide permeation against the background of the minimal myocardial creatinine release. In this group there was also a decrease in Ca2+ loss at the beginning of reperfusion (immediately after ischemia). The above phenomenon explains the least degree of ischemic myocardial damage in the isolated mouse heart. The obtained data should be expanded. Since it is difficult to accurately determine the dose of carbon monoxide, then the use of donor compounds is promising. Such compounds include CORM-2 and CORM-3. Under physiological conditions, they decompose in a controlled manner, releasing a specific amount of carbon monoxide. Conclusion. The obtained results indicate that at different concentrations of carbon monoxide can differently influence different structures of cardiomyocyte: at one concentration it binds to calcium channels, other concentrations influence ion channels of plasma membrane, which can explain all these dependencies

Common Genetic Variants Contribute to Risk of Transposition of the Great Arteries

Background: Dextro-transposition of the great arteries (D-TGA) is a severe congenital heart defect which affects approximately 1 in 4,000 live births. While there are several reports of D-TGA patients with rare variants in individual genes, the majority of D-TGA cases remain genetically elusive. Familial recurrence patterns and the observation that most cases with D-TGA are sporadic suggest a polygenic inheritance for the disorder, yet this remains unexplored. Methods: We sought to study the role of common single nucleotide polymorphisms (SNPs) in risk for D-TGA. We conducted a genome-wide association study in an international set of 1,237 patients with D-TGA and identified a genome-wide significant susceptibility locus on chromosome 3p14.3, which was subsequently replicated in an independent case-control set (rs56219800, meta-analysis P=8.6x10-10, OR=0.69 per C allele). Results: SNP-based heritability analysis showed that 25% of variance in susceptibility to D-TGA may be explained by common variants. A genome-wide polygenic risk score derived from the discovery set was significantly associated to D-TGA in the replication set (P=4x10-5). The genome-wide significant locus (3p14.3) co-localizes with a putative regulatory element that interacts with the promoter of WNT5A, which encodes the Wnt Family Member 5A protein known for its role in cardiac development in mice. We show that this element drives reporter gene activity in the developing heart of mice and zebrafish and is bound by the developmental transcription factor TBX20. We further demonstrate that TBX20 attenuates Wnt5a expression levels in the developing mouse heart. Conclusions: This work provides support for a polygenic architecture in D-TGA and identifies a susceptibility locus on chromosome 3p14.3 near WNT5A. Genomic and functional data support a causal role of WNT5A at the locus.

NRSF-GNAO1 Pathway Contributes to the Regulation of Cardiac Ca 2+ Homeostasis

Background: During the development of heart failure, a fetal cardiac gene program is reactivated and accelerates pathological cardiac remodeling. We previously reported that a transcriptional repressor, neuron restrictive silencer factor (NRSF), suppresses the fetal cardiac gene program, thereby maintaining cardiac integrity. The underlying molecular mechanisms remains to be determined, however. Methods: We aim to elucidate molecular mechanisms by which NRSF maintains normal cardiac function. We generated cardiac-specific NRSF knockout mice and analyzed cardiac gene expression profiles in those mice and mice cardiac-specifically expressing a dominant-negative NRSF mutant. Results: We found that cardiac expression of Gαo, an inhibitory G protein encoded in humans by GNAO1, is transcriptionally regulated by NRSF and is increased in the ventricles of several mouse models of heart failure. Genetic knockdown of Gnao1 ameliorated the cardiac dysfunction and prolonged survival rates in these mouse heart failure models. Conversely, cardiac-specific overexpression of GNAO1 in mice was sufficient to induce cardiac dysfunction. Mechanistically, we observed that increasing Gαo expression increased surface sarcolemmal L-type Ca 2+ channel activity, activated Calcium/calmodulin-dependent kinase-II (CaMKII) signaling and impaired Ca 2+ handling in ventricular myocytes, which led to cardiac dysfunction. Conclusions: These findings shed light on a novel function of Gαo in the regulation of cardiac Ca 2+ homeostasis and systolic function and suggest Gαo may be an effective therapeutic target for the treatment of heart failure.

Ferulic Acid Alleviates Oxidative Stress-Induced Cardiomyocyte Injury by the Regulation of miR-499-5p/p21 Signal Cascade

Objective. To investigate the protective effects and regulatory mechanisms of ferulic acid on oxidative stress-induced cardiomyocyte injury. Methods. We established a cardiomyocyte oxidative stress cell model by H2O2 treatment and a mouse heart injury model by isoprenaline infusion of male C57BL/6 mice. Ferulic acid was applied to treat oxidative stress-induced cardiomyocyte injury. DHE staining was used to detect ROS production. DNA fragmentation, TUNEL assay, and cleaved caspase-3 were used to analyze cell apoptosis. Real-time PCR and Western blotting were used to analyze miRNA and protein levels to investigate the regulatory mechanisms of ferulic acid on oxidative stress-induced cardiomyocyte injury. Results. Ferulic acid pretreatment significantly inhibited H2O2- and isoprenaline-induced oxidative stress and cell apoptosis by promoting miR-499-5p expression and inhibiting p21 expression. MiR-499-5p inhibition reversed the protective effects of ferulic acid. Further study found that ferulic acid could also attenuate isoprenaline-induced mouse heart fibrosis and cell apoptosis by reducing oxidative stress, inflammation, and apoptosis in vivo. Conclusions. We proved that ferulic acid protects cardiomyocytes from oxidative stress-induced injury by regulating the miR-499-5p/p21signaling pathway, which provides insight into the clinical application of ferulic acid in the treatment of cardiovascular diseases.