Neuroprotective peptide OL-FS13 attenuates cardiac apoptosis and myocardial infarction injury through Nrf2/HO-1 pathway

High incidence of cardiac rupture in murine myocardial infarction (MI) model leads to a substantial loss before the study end-point. Selecting animal models with varying degrees of injury for different research purposes is crucial for cardiovascular research. Male C57 mice were subjected to ischemia/reperfusion (I/R) or permanent occlusion (MI) injury. The incidence of cardiac rupture, degree of myocardial injury, inflammatory responses, left ventricular (LV) remodeling and infarct myocardium healing were examined. Compared to MI mice, early reperfusion (1, 2 and 4h I/R) completely prevented cardiac rupture, while delayed reperfusion (12h and 24h I/R) significantly reduced incidence of cardiac rupture to 5.7% and 8.6%, respectively. In the acute phase, prolonged ischemia increased infarct size, myocyte apoptosis, and both systemic and regional inflammatory responses. These changes correspond to enhanced MMP-9 activity and a weakening of the tensile strength of the infarcted myocardium. Following ischemic insult, early reperfusion was associated with less extent of myocardial injury, inflammatory response and adverse cardiac remodeling, whereas, delayed reperfusion and MI groups exhibited severe myocardial damage and remodeling. Furthermore, both early and delayed reperfusion were associated with increased infiltration of type 2 macrophages and proliferation of endothelial cells during the early healing phase, thereby facilitating healing of the infarct myocardium. Delayed reperfusion resulted in a comparable and substantial degree of cardiac remodeling but with a lower risk of cardiac rupture in comparison with MI model. This feature makes it a feasible model for cardiac ischemia research.

Many novel therapies to treat myocardial infarction (MI), yielding promising results in animal models, nowadays failed in clinical trials for several reasons. The most used animal MI model is based on permanent ligation of the left anterior descending (LAD) coronary artery in healthy mice resulting in transmural MI, while in clinical practice reperfusion is usually accomplished by primary percutaneous coronary interventions (PCI) limiting myocardial damage and inducing myocardial ischemia–reperfusion (MI-R) injury. To evaluate a more similar murine MI model we compared MI-R injury to unreperfused MI in hypercholesterolemic apolipoprotein (APO)E*3-Leiden mice regarding effects on cardiac function, left ventricular (LV) remodeling and inflammation. Both MI-R and MI resulted in significant LV dilation and impaired cardiac function after 3 weeks. Although LV dilation, displayed by end-diastolic (EDV) and end-systolic volumes (ESV), and infarct size (IS) were restricted following MI-R compared to MI (respectively by 27.6% for EDV, 39.5% ESV, 36.0% IS), cardiac function was not preserved. LV-wall thinning was limited with non-transmural LV fibrosis in the MI-R group (66.7%). Two days after inducing myocardial ischemia, local leucocyte infiltration in the infarct area was decreased following MI-R compared to MI (36.6%), whereas systemic circulating monocytes were increased in both groups compared to sham (130.0% following MI-R and 120.0% after MI). Both MI-R and MI models against the background of a hypercholesterolemic phenotype appear validated experimental models, however reduced infarct size, restricted LV remodeling as well as a different distributed inflammatory response following MI-R resemble the contemporary clinical outcome regarding primary PCI more accurately which potentially provides better predictive value of experimental therapies in successive clinical trials.

BackgroundExtracellular vesicles (EVs) derived from various cell sources exert cardioprotective effects during cardiac ischemic injury. Our previous study confirmed that EVs derived from ischemic-reperfusion injured heart tissue aggravated cardiac inflammation and dysfunction. However, the role of EVs derived from normal cardiac tissue in myocardial ischemic injury remains elusive.ResultsIn the present study, normal heart-derived EVs (cEVs) and kidney-derived EVs (nEVs) were isolated and intramyocardially injected into mice after myocardial infarction (MI). We demonstrated that administration of both cEVs and nEVs significantly improved cardiac function, reduced the scar size, and alleviated inflammatory infiltration into the heart. In addition, cardiomyocyte apoptosis was inhibited, whereas angiogenesis was enhanced in the hearts receiving cEVs or nEVs treatment. Moreover, intramyocardial injection of cEVs displayed much better cardiac protective efficacy than nEVs in murine MI models. RNA-seq and protein-protein interaction (PPI) network analysis revealed the protective mRNA clusters in both cEVs and nEVs. These mRNAs were involved in multiple signaling pathways, which may synergistically orchestrate to prevent the heart from further damage post MI.ConclusionsCollectively, our results indicated that EVs derived from normal heart tissue may represent a promising strategy for cardiac protection in ischemic heart diseases.

In healthy hearts, tumor necrosis factor-α (TNF-α) concentration is low, and TNF-α is mainly located in endothelium and resident mast cells.1 TNF-α receptors (TNFRs) 1 (TNFR1) and 2 (TNFR2) are expressed on most cardiac cells, including cardiomyocytes.2 During myocardial ischemia, preformed TNF-α is released within minutes from resident mast cells and macrophages.3 With persistent ischemia, TNF-α also originates from cardiomyocytes.4 In rats after myocardial infarction (MI), TNFR1 density is increased for 10 days, whereas TNFR2 density remains unchanged.5 In contrast, TNFR1 and TNFR2 are both downregulated in the failing heart, whereas soluble TNFRs are increased6 owing to proteolytic cleavage of cardiomyocyte TNFR and release from circulating exosome-like vesicles.7 Increased soluble TNFRs decrease TNF-α bioactivity while at the same time prolonging its half-life.8 Article p 1386 TNF-α contributes to both reversible (contractile dysfunction4,9) and irreversible (MI) injury.1 Preischemic treatment with TNF-α antibodies10 or soluble TNFR1,11 permanent TNF-α knockout,12 or knockout of TNFR1 but not TNFR213 all reduce infarct size. The latter finding highlights the functional difference of TNFR1 and TNFR2 activation for ischemia/reperfusion injury: Only TNFR1 signaling is detrimental (Figure). Figure. Signaling cascade activated (→) or inhibited (⊣) by TNFR1 (red) or TNFR2 (blue) activation. sTNFR1 indicates soluble TNFR1; sTNFR2, soluble TNFR2; S1-P, sphingosine-1-phosphate; NF-κB, nuclear factor-κB; SOCS3, suppressor of cytokine signaling 3; ROS, reactive oxygen species; Akt-P, phosphorylated Akt, a homologue of the transforming v-Akt; NADPH-Ox, NADPH oxidase; STAT3-P, phosphorylated signal transducer and activator of transcription; Jnk-P, phosphorylated c-jun N-terminal kinase; p38-P, phosphorylated mitogen-activated protein kinase; Erk-P, phosphorylated extracellular signal-regulated kinase; PKC, protein kinase C; CamK, calcium/calmodulin-dependent kinase; MMP-2/9, matrix metalloproteinases 2 and 9; and IL-6, interleukin 6. Red indicates proteins involved in apoptosis; purple, proteins involved in growth; green, …

Cortical bone stem cells (CBSCs) have been shown to reduce ventricular remodeling and improve cardiac function in a murine myocardial infarction (MI) model. These effects were superior to other stem cell types that have been used in recent early-stage clinical trials. However, CBSC efficacy has not been tested in a preclinical large animal model using approaches that could be applied to patients. To determine whether post-MI transendocardial injection of allogeneic CBSCs reduces pathological structural and functional remodeling and prevents the development of heart failure in a swine MI model. Female Göttingen swine underwent left anterior descending coronary artery occlusion, followed by reperfusion (ischemia-reperfusion MI). Animals received, in a randomized, blinded manner, 1:1 ratio, CBSCs (n=9; 2×107 cells total) or placebo (vehicle; n=9) through NOGA-guided transendocardial injections. 5-ethynyl-2'deoxyuridine (EdU)-a thymidine analog-containing minipumps were inserted at the time of MI induction. At 72 hours (n=8), initial injury and cell retention were assessed. At 3 months post-MI, cardiac structure and function were evaluated by serial echocardiography and terminal invasive hemodynamics. CBSCs were present in the MI border zone and proliferating at 72 hours post-MI but had no effect on initial cardiac injury or structure. At 3 months, CBSC-treated hearts had significantly reduced scar size, smaller myocytes, and increased myocyte nuclear density. Noninvasive echocardiographic measurements showed that left ventricular volumes and ejection fraction were significantly more preserved in CBSC-treated hearts, and invasive hemodynamic measurements documented improved cardiac structure and functional reserve. The number of EdU+ cardiac myocytes was increased in CBSC- versus vehicle- treated animals. CBSC administration into the MI border zone reduces pathological cardiac structural and functional remodeling and improves left ventricular functional reserve. These effects reduce those processes that can lead to heart failure with reduced ejection fraction.

Buyang Huanwu decoction inhibits cardiomyocyte apoptosis after myocardial infarction by enhancing aldehyde dehydrogenase-2 activity and protein expression

Introduction: Ischemic heart disease is the leading cause of death in adults in the US. We have shown that overexpression of E3 ligase Pellino1 (Peli1) increased vessel density, triggered anti-apoptotic signaling, and preserved cardiac function in the infarcted myocardium. In the present study, we aimed to explore whether the loss of Peli1 function in cardiomyocytes has any effect on cardiac function using a genetically-modified murine myocardial infarction (MI) model. Methods: Wild type (WT) and cardiomyocyte-specific Peli1 knockout (CP1KO -/- ) mice were divided into 4 groups [wild-type-sham (WTS), CP1KO -/- sham (CP1KO -/- S), WTMI and CP1KO -/- MI] to undergo either left anterior descending artery ligation or sham surgery. Phosphorylation of Mitogen-Activated Protein Kinase-Activated Protein Kinase-2 (p-MK2) and expression of VEGF, Bcl2, and cIAP2 were quantified by Western blot analysis. Cardiac function by two-dimensional echocardiography was performed at 30 days post-op to assess ejection fraction (EF), fractional shortening (FS), left ventricular internal diameter in both systole and diastole (LVIDs and LVIDd). Results: We observed no significant change in cardiac functions between WTS and CP1KO-/-S group. However, in MI groups, CP1KO -/- mice showed significant loss of systolic functions, represented by both EF (26.60%±2.340 vs. 33.46%±1.84; p<0.029, n=13) and FS (12.43%± 1.19 vs. 15.93%±0.98; n=13, p<0.03) when compared to WTMI group. Western blot analysis of heart tissue 24-hour post-MI showed a significant reduction in the expression of p-MK2 (p=0.0045) in CP1KO -/- group compared to WT mice. Similarly, expression of VEGF (p=0.0227), BCl-2 (p=0.0073) and cIAP2 (p=0.0483) was also found to be significantly downregulated in CP1KO -/- group compared to WTMI group at 4-day post-MI. We also analyzed oxidative stress marker, 3-NT, which was significantly upregulated (p=0.0001) in CP1KO mice compared to WTMI. Conclusion: Loss of Peli1 in cardiomyocyte results in the impairment of cardiac function along with increased oxidative stress, reduced angiogenic and survival protein expression, which suggests Peli1 plays an important role in the cardiomyocyte signaling during myocardial infarction.

The microenvironment of native heart tissue may be better replicated when cardiomyocytes are cultured in three-dimensional clusters (i.e., spheroids) than in monolayers or as individual cells. Thus, we differentiated human cardiac lineage-induced pluripotent stem cells in cardiomyocytes (hiPSC-CMs) and allowed them to form spheroids and spheroid fusions that were characterized in vitro and evaluated in mice after experimentally induced myocardial infarction (MI). Synchronized contractions were observed within 24 h of spheroid formation, and optical mapping experiments confirmed the presence of both Ca2+ transients and propagating action potentials. In spheroid fusions, the intraspheroid conduction velocity was 7.0 ± 3.8 cm/s on days 1- 2 after formation, whereas the conduction velocity between spheroids increased significantly ( P = 0.003) from 0.8 ± 1.1 cm/s on days 1- 2 to 3.3 ± 1.4 cm/s on day 7. For the murine MI model, five-spheroid fusions (200,000 hiPSC-CMs/spheroid) were embedded in a fibrin patch and the patch was transplanted over the site of infarction. Later (4 wk), echocardiographic measurements of left ventricular ejection fraction and fractional shortening were significantly greater in patch-treated animals than in animals that recovered without the patch, and the engraftment rate was 25.6% or 30% when evaluated histologically or via bioluminescence imaging, respectively. The exosomes released from the spheroid patch seemed to increase cardiac function. In conclusion, our results established the feasibility of using hiPSC-CM spheroids and spheroid fusions for cardiac tissue engineering, and, when fibrin patches containing hiPSC-CM spheroid fusions were evaluated in a murine MI model, the engraftment rate was much higher than the rates we have achieved via the direct intramyocardial injection. NEW & NOTEWORTHY Spheroids fuse in culture to produce structures with uniformly distributed cells. Furthermore, human cardiac lineage-induced pluripotent stem cells in cardiomyocytes in adjacent fused spheroids became electromechanically coupled as the fusions matured in vitro, and when the spheroids were combined with a biological matrix and administered as a patch over the infarcted region of mouse hearts, the engraftment rate exceeded 25%, and the treatment was associated with significant improvements in cardiac function via a paracrine mechanism, where exosomes released from the spheroid patch.

Background: Myocardial infarction (MI) is a leading cause of heart failure. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) show therapeutic potential for MI, largely attributed to paracrine effects. However, the role of microvesicles (MVs), a major class of extracellular vesicles, n this effect remains unclear. Therefore, it is imperative to ascertain whether hiPSC-CM derived MVs exhibit cardioprotective effects and to elucidate the underlying mechanisms in both murine and porcine models of MI. Methods: hiPSC-CM-MVs were isolated by differential ultracentrifugation and characterized via nanoparticle tracking, electron microscopy, flow cytometry, and western blot. Their therapeutic effects were evaluated in oxygen–glucose deprivation (OGD)-injured hiPSC-CMs and murine MI models. Proteomic profiling identified candidate effectors, which were further validated using cardiac-specific gene overexpression or knockout transgenic mouse models. A porcine MI model was also used to assess the safety and efficacy of MV through MDCT, hemodynamic measurements, and histological analysis. Results: In vitro, we initially isolated high-purity MVs from hiPSC-CMs, exhibiting a distinct enrichment of mitochondrial constituents.These MVs conferred robust cytoprotection against OGD-induced injury by suppressing cardiomyocyte apoptosis, augmenting mitochondrial membrane potential and calcium uptake capacity, and thereby sustaining calcium homeostasis. In vivo, the trentment of MV alleviated progressive cardiac dysfunction following MI in mice and concurrently suppressed a spectrum of pathological remodeling events, including myocardial hypertrophy, fibrosis, apoptosis, and calcium overload. The cardioprotective effects observed were likely the result of enhanced mitochondrial calcium uptake, which contributed to restore calcium homeostasis. To explore the underlying mechanism, proteomic profiling of MVs identified DExH-box helicase 9 (DHX9) as a key regulatory cargo. Cardiac-specific overexpression of DHX9 partially reproduced while knockout DHX9 abolished the effects of MVs. Finally, in a porcine MI model, MVs exhibited robust therapeutic efficacy and safety without systemic toxicity. Conclusion: This study identifies a novel mechanism by which hiPSC-CM-MVs restore calcium homeostasis and improve cardiac function post-MI via DHX9. These findings provide preclinical evidence supporting MVs as a safe and effective therapeutic strategy for MI.

The clinical efficacy of neuregulin (NRG) in the treatment of heart failure is hindered by off-target exposure due to systemic delivery. We previously encapsulated neuregulin in a hydrogel (HG) for targeted and sustained myocardial delivery, demonstrating significant induction of cardiomyocyte proliferation and preservation of post-infarct cardiac function in a murine myocardial infarction (MI) model. Here, we performed a focused evaluation of our hydrogel-encapsulated neuregulin (NRG-HG) therapy’s potential to enhance cardiac function in an ovine large animal MI model. Adult male Dorset sheep (n = 21) underwent surgical induction of MI by coronary artery ligation. The sheep were randomized to receive an intramyocardial injection of saline, HG only, NRG only, or NRG-HG circumferentially around the infarct borderzone. Eight weeks after MI, closed-chest intracardiac pressure–volume hemodynamics were assessed, followed by heart explant for infarct size analysis. Compared to each of the control groups, NRG-HG significantly augmented left ventricular ejection fraction (p = 0.006) and contractility based on the slope of the end-systolic pressure–volume relationship (p = 0.006). NRG-HG also significantly reduced infarct scar size (p = 0.002). Overall, using a bioengineered hydrogel delivery system, a one-time dose of NRG delivered intramyocardially to the infarct borderzone at the time of MI in adult sheep significantly reduces scar size and enhances ventricular contractility at 8 weeks after MI.

Plasma IL-6 is elevated after myocardial infarction (MI) and is associated with increased morbidity and mortality. Which cardiac cell type preferentially contributes to IL-6 expression and how its production is regulated are largely unknown. Here, we studied the cellular source and purinergic regulation of IL-6 formation in a murine MI model. We found that IL-6, measured in various cell types in post-MI hearts at the protein level and by quantitative PCR and RNAscope, was preferentially formed by cardiac fibroblasts (CFs). Single-cell RNA-Seq (scRNA-Seq) in infarcted mouse and human hearts confirmed this finding. We found that adenosine stimulated fibroblast IL-6 formation via the adenosine receptor A2bR in a Gq-dependent manner. CFs highly expressed Adora2b and rapidly degraded extracellular ATP to AMP but lacked CD73. In mice and humans, scRNA-Seq revealed that Adora2B was also mainly expressed by fibroblasts. We assessed global IL-6 production in isolated hearts from mice lacking CD73 on T cells (CD4-CD73–/–), a condition known to be associated with adverse cardiac remodeling. The ischemia-induced release of IL-6 was strongly attenuated in CD4-CD73–/– mice, suggesting adenosine-mediated modulation. Together, these findings demonstrate that post-MI IL-6 was mainly derived from activated CFs and was controlled by T cell–derived adenosine. We show that purinergic metabolic cooperation between CFs and T cells is a mechanism that modulates IL-6 formation by the heart and has therapeutic potential.

Ongoing basic molecular analyses are being performed in mice, and a simple long-surviving murine model of myocardial infarction (MI) would be very useful in this regard. Although a few studies have induced MI in mice by coronary artery ligation, the induction involves a complex technique and has a relatively high mortality rate. In addition, the identification of the basic pathological sequence is essential to the interpretation of experimental results. We developed a simple technique for the induction of MI in mice and examined qualitative and quantitative conventional microscopic findings during the pathological evolution over a 28-day observation period. Male BALB/c mice weighing approximately 25-30 g were anesthetized and then ventilated with a positive pressure ventilator. The heart was exposed by thoracotomy. Left coronary artery occlusion was performed by thermocoagulation using a thermocoagulation knife at the level of the tip of the left atrium. After establishing this surgical method, we used it to induce MI in 71 mice. The operative and postoperative mortality rates of this model were 5.6% (4/71) and 12.6% (9/71), respectively. In 3 (5.2%) of the 58 surviving mice, the area of infarct was not sufficient. The infarct area in the remaining 55 mice was 40 +/- 9% of the entire perimeter of the left ventricle. Conventional microscopic examinations with hematoxylin-eosin and Masson-trichrome staining disclosed that all of the characteristic histopathological features of MI occurred 1-2 days earlier than those in rats. Our surgical technique provides a sufficient infarct area, with an acceptable mortality rate. The present study clarified the histopathological sequence in this long surviving murine MI model.

Background: Myelomonocytic cells are involved in both the initial injury phase and the healing process of myocardial infarction (MI). However, the exact interaction of inflammatory myeloid cells and prominent cytokines remains only partly understood. Objective: The goal of the study was to investigate the either cardioprotective or in part adverse role of lysozyme M positive (LysM+) and granulocyte-receptor 1 positive (Gr-1+) immune cells on cardiac injury and healing in a murine model of MI. Methods and results: MI was induced in 8 to 12 week-old male mice (C57BL/6 background) by ligation of the left anterior descending (LAD) coronary artery. Compared to LysMCre controls, LysM+ cell depleted LysMiDTR transgenic mice (depletion 3d prior MI by diphtheria toxin application, 25 ng/g body weight) showed a decreased influx of CD45.2+/CD3-/CD11b+/Gr-1high neutrophils into infarcted myocardium 1d post MI (measured by flow cytometric analysis). Additionally cardiac mRNA expression levels of inflammatory cytokines like INFγ and TNFα were decreased 7d post MI. To more specifically assess the role of neutrophils, we depleted C57BL/6 mice with a monoclonal anti-Gr-1 antibody and found increased mortality early after MI as well as a decrease in INFγ mRNA expression 1d and 7d post MI. MCP-1 (CCL2) and CCR2 mRNA were reduced 3d after MI according to decreased amount of CD11b+/Ly6G-/Ly6Chigh inflammatory monocytes in the infarcted myocardium of anti-Gr-1 treated mice. LAD ligated INFγ-/- mice displayed a significantly reduced survival, worsening of left ventricular function and an impaired inflammatory cell infiltration compared to C57BL/6 controls. Conclusion: We provide evidence that neutrophils and INFγ play an essential role in survival and cardiac remodeling following MI. Our data indicate that neutrophils are required for monocyte chemotaxis. We conclude, that strategies to combat the inflammatory injury in MI must consider a potentially beneficial effect of early neutrophil influx into infarcted myocardium.

Ischemic heart disease and subsequent myocardial infarction (MI) is one of the leading causes of mortality in the United States and around the world. In order to explore the pathophysiological changes after myocardial infarction and design future treatments, research models of MI are required. Permanent ligation of the left coronary artery (LCA) in mice is a popular model to investigate cardiac function and ventricular remodeling post MI. Here we describe a less invasive, reliable, and reproducible surgical murine MI model by permanent ligation of the LCA. Our surgical model comprises of an easily reversible general anesthesia, endotracheal intubation that does not require a tracheotomy, and a thoracotomy. Electrocardiography and troponin measurement should be performed to ensure MI. Echocardiography at day 28 after MI will discern heart function and heart failure parameters. The degree of cardiac fibrosis can be evaluated by Masson's trichrome staining and cardiac MRI. This MI model is useful for studying the pathophysiological and immunological alterations after MI.

Introduction Despite progress in pharmacological treatment of myocardial infarction (MI), there is still an immense need for novel therapies for this life-threatening condition. Accordingly, cell-based therapies have been extensively investigated with most studies focusing on mesenchymal stromal cells. However due to their inability to differentiate into cardiomyocytes as well as limited survival upon in vivo administration, no effective treatment of MI has been developed. In contrast, application of hiPSC-derived cardiomyocytes (hiPSC-CM) represent biologically rational approach with pre-clinical studies confirming their therapeutic potential in various models of MI. However further optimization is required due to limited survival of hiPSC-CM upon in vivo administration. Therefore, we evaluated the therapeutic potential of genetically modified hiPSC-CM in murine model of acute MI and compared it to the effect of adipose tissue-derived stromal cells (ADSC). Methods In the first step hiPSC overexpressing GFP, luciferase (Luc) and pro-angiogenic and cardioprotective factors: heme oxygenase-1 (HO-1, heme degrading enzyme) or stromal cell-derived factor-1 (SDF-1, pro-angiogenic chemokine) were subjected to cardiac differentiation which yielded in each group 70–90% cardiac troponin T-positive contracting cells. hiPSC-CM (5x105 in 10 μl) were administered into NOD-SCID mice which underwent permanent ligation of left anterior descending (LAD) coronary artery. The cells were injected into the peri-infarct zone. Mice subjected to sham operation as well as injected with saline after MI were used as controls. The ultrasonography of hearts was performed on day 7, 14, 28 and 42 whereas the presence of hiPSC-CM was monitored using IVIS Spectrum system upon administration of luciferin and analysed in sections of collected hearts. The same experimental scheme was used to assess therapeutic potential of ADSC (CD105+CD73+CD90+CD44+CD146-CD34-) overexpressing luciferase and GFP. Results Ultrasonography demonstrated that upon delivery of hiPSC-CM the left ventricle ejection fraction (LVEF) was very significantly higher in comparison to control group injected with saline after induction of MI. In contrast, no improvement of LVEF was observed after administration of ADSC. Interestingly, measurements of luciferase activity revealed the strongest bioluminescent signal in the hearts of mice transplanted with iPSC-CM-HO1 42 days after MI. Importantly, the survival of hiPSC-CM in murine myocardium six weeks upon administration was further confirmed with immunofluorescent analysis of heart sections using human specific anti-Ku80 antibody. Again, luciferase activity was not observed upon delivery of ADSC. Conclusion These results strongly indicate that administration of hiPSC-CM, unlike ADSC, preserve murine heart function in acute MI model. Additionally, overexpression of HO-1 may positively influence their survival upon in vivo delivery into infarcted tissue. Acknowledgement/Funding The National Centre for Research and Development (STRATEGMED 2/269415/11/NCBR/2015), National Science Centre of Poland (HARMONIA 2014/14/M/NZ1/00010)