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Background & Aims: Neuroinflammation in the gut is associated with many gastrointestinal (GI) diseases, including inflammatory bowel disease. In the brain, neuroinflammatory conditions are associated with blood-brain barrier (BBB) disruption and subsequent neuronal injury. We sought to determine whether the enteric nervous system is similarly protected by a physical barrier and whether that barrier is disrupted in colitis. Methods: Confocal and electron microscopy were used to characterize myenteric plexus structure, and FITC-dextran assays were used to assess for presence of a barrier. Colitis was induced with dextran sulfate sodium, with co-administration of liposome-encapsulated clodronate to deplete macrophages. Results: We identified a blood-myenteric barrier (BMB) consisting of extracellular matrix proteins (agrin and collagen-4) and glial end-feet, reminiscent of the BBB, surrounded by a collagen-rich periganglionic space. The BMB is impermeable to the passive movement of 4 kDa FITC-dextran particles. A population of macrophages is present within enteric ganglia (intraganglionic macrophages [IGMs]) and exhibits a distinct morphology from muscularis macrophages, with extensive cytoplasmic vacuolization and mitochondrial swelling but without signs of apoptosis. IGMs can penetrate the BMB in physiological conditions and establish direct contact with neurons and glia. Dextran sulfate sodium-induced colitis leads to BMB disruption, loss of its barrier integrity, and increased numbers of IGMs in a macrophage-dependent process. Conclusions: In intestinal inflammation, macrophage-mediated degradation of the BMB disrupts its physiological barrier function, eliminates the separation of the intra- and extra-ganglionic compartments, and allows inflammatory stimuli to access the myenteric plexus. This suggests a potential mechanism for the onset of neuroinflammation in colitis and other GI pathologies with acquired enteric neuronal dysfunction.

Summary: Excessive binge alcohol drinking may adversely affect cardiovascular function. In this study we characterize the detailed hemodynamic effects of an acute alcohol binge in mice using multiple approaches and investigate the role of the endocannabinoid–cannabinoid 1 receptor (CB1-R) signaling in these effects. Acute alcohol binge was associated with elevated levels of cardiac endocannabinoid anandamide and profound cardiovascular dysfunction lasting for several hours and redistribution of circulation. These changes were attenuated by CB1-R antagonist or in CB1-R knockout mice. Our results suggest that a single alcohol binge has profound effects on the cardiovascular system, which involve endocannabinoid–CB1-R signaling. Key Words: binge alcohol drinking, cannabinoids, contractility, endocannabinoids

Extensive research work has been carried out to define the exact significance and contribution of regulated necrosis-like cell death program, such as necroptosis to cardiac ischemic injury. This cell damaging process plays a critical role in the pathomechanisms of myocardial infarction (MI) and post-infarction heart failure (HF). Accordingly, it has been documented that the modulation of key molecules of the canonical signaling pathway of necroptosis, involving receptor-interacting protein kinases (RIP1 and RIP3) as well as mixed lineage kinase domain-like pseudokinase (MLKL), elicit cardioprotective effects. This is evidenced by the reduction of the MI-induced infarct size, alleviation of myocardial dysfunction, and adverse cardiac remodeling. In addition to this molecular signaling of necroptosis, the non-canonical pathway, involving Ca2+/calmodulin-dependent protein kinase II (CaMKII)-mediated regulation of mitochondrial permeability transition pore (mPTP) opening, and phosphoglycerate mutase 5 (PGAM5)–dynamin-related protein 1 (Drp-1)-induced mitochondrial fission, has recently been linked to ischemic heart injury. Since MI and HF are characterized by an imbalance between reactive oxygen species production and degradation as well as the occurrence of necroptosis in the heart, it is likely that oxidative stress (OS) may be involved in the mechanisms of this cell death program for inducing cardiac damage. In this review, therefore, several observations from different studies are presented to support this paradigm linking cardiac OS, the canonical and non-canonical pathways of necroptosis, and ischemia-induced injury. It is concluded that a multiple therapeutic approach targeting some specific changes in OS and necroptosis may be beneficial in improving the treatment of ischemic heart disease.

Hepatic infiltration of neutrophils is a hallmark of steatohepatitis; however, the role of neutrophils in the progression of steatohepatitis remains unknown. Methods: A clinically relevant mouse model of steatohepatitis induced by high-fat diet (HFD) plus binge ethanol feeding was used. Liver fibrosis was examined. In vitro cell culture was used to analyze the interaction of hepatic stellate cells (HSCs) and neutrophils. Results: HFD plus one binge ethanol (HFD+1B) feeding induced significant hepatic neutrophil infiltration, liver injury, and fibrosis. HFD plus multiple binges of ethanol (HFD+mB) caused more pronounced liver fibrosis. Microarray analyses showed that the most highly activated signaling pathway in this HFD+1B model was related to liver fibrosis and HSC activation. Blockade of chemokine (C-X-C motif) ligand 1 or intercellular adhesion molecule-1 expression reduced hepatic neutrophil infiltration and ameliorated liver injury and fibrosis. Disruption of the p47phox gene (also called neutrophil cytosolic factor 1), a critical component of reactive oxygen species producing nicotinamide adenine dinucleotide phosphate-oxidase in neutrophils, diminished HFD+1B–induced liver injury and fibrosis. Co-culture of HSCs with neutrophils, but not with neutrophil apoptotic bodies, induced HSC activation and prolonged neutrophil survival. Mechanistic studies showed that activated HSCs produce granulocyte-macrophage colony-stimulating factor and interleukin-15 to prolong the survival of neutrophils, which may serve as a positive forward loop to promote liver damage and fibrosis. Conclusions: The current data from a mouse model of HFD plus binge ethanol feeding suggest that obesity and binge drinking synergize to promote liver fibrosis, which is partially mediated via the interaction of neutrophils and HSCs. Microarray data in this article have been uploaded to NCBI’s Gene Expression Omnibus (GEO accession number: GSE98153).

Cardiovascular disease (CVD) remains the leading cause of death worldwide and, despite continuous advances, better diagnostic and prognostic tools, as well as therapy, are needed. The human transcriptome, which is the set of all RNA produced in a cell, is much more complex than previously thought and the lack of dialogue between researchers and industrials and consensus on guidelines to generate data make it harder to compare and reproduce results. This European Cooperation in Science and Technology (COST) Action aims to accelerate the understanding of transcriptomics in CVD and further the translation of experimental data into usable applications to improve personalized medicine in this field by creating an interdisciplinary network. It aims to provide opportunities for collaboration between stakeholders from complementary backgrounds, allowing the functions of different RNAs and their interactions to be more rapidly deciphered in the cardiovascular context for translation into the clinic, thus fostering personalized medicine and meeting a current public health challenge. Thus, this Action will advance studies on cardiovascular transcriptomics, generate innovative projects, and consolidate the leadership of European research groups in the field. COST (European Cooperation in Science and Technology) is a funding organization for research and innovation networks (www.cost.eu).

Abstract Aims The association and co-isolation of low-density lipoproteins (LDL) and extracellular vesicles (EVs) have been shown in blood plasma. Here we explore this relationship to better understand the role of EVs in atherogenesis. Methods and results Wild type (WT), PCSK9−/−, and LDLR−/− C57BL/6 mice were used in this study. Eleven week-old male mice were fed high-fat diet (HFD) for 12 weeks or kept on normal diet until old age (22-months). Cardiac function was assessed by ultrasound, cholesterol was quantified with a colorimetric kit and circulating EVs were measured using flow cytometry. Plaques were analysed post-mortem using Oil-Red-O staining of the aortic arch. EVs were measured from platelet free blood plasma samples of normal and hypercholesterolaemic clinical patients. Based on annexin V and CD63 staining, we found a significant increase in EV levels in LDLR−/− and PCSK9−/− mice after HFD, but CD81 showed no significant change in either group. There was no significant change in plaque formation after HFD. PCSK9−/− mice show a favourable cardiac function after HFD. Blood cholesterol levels progressively increased during HFD, with LDLR−/− mice showing high levels while PCSK9−/− were significantly lowered compared to WT animals. In mice at old age, similar cholesterol levels were observed as in young mice. In old age, LDLR−/− mice showed significantly increased plaques. At old age, ejection fraction was decreased in all groups of mice, as were CD63+ EVs. Similarly to mice, in patients with hypercholesterolaemia, CD63+ EVs were significantly depleted. Conclusions This research demonstrates an inverse relationship between circulating EVs and cholesterol, making EVs a potential marker for cardiovascular disease (CVD). HFD causes reduced cardiac function, but atherosclerotic development is slowly progressing in hypercholesterolaemic models and only observed with old animals. These results also bring further evidence for the benefit of using of PCSK9 inhibitors as therapeutic agents in CVD.

Abstract Previously, the presence of a blood-myenteric plexus barrier and its disruption was reported in experimentally induced colitis via a macrophage-dependent process. The aim of this study is to reveal how myenteric barrier disruption and subsequent neuronal injury affects gut motility in vivo in a murine colitis model. We induced colitis with dextran sulfate sodium (DSS), with the co-administration of liposome-encapsulated clodronate (l-clodronate) to simultaneously deplete blood monocytes contributing to macrophage infiltration in the inflamed muscularis of experimental mice. DSS-treated animals receiving concurrent l-clodronate injection showed significantly decreased blood monocyte numbers and colon muscularis macrophage (MM) density compared to DSS-treated control (DSS-vehicle). DSS-clodronate-treated mice exhibited significantly slower whole gut transit time than DSS-vehicle-treated animals and comparable to that of controls. Experiments with oral gavage-fed Evans-blue dye showed similar whole gut transit times in DSS-clodronate-treated mice as in control animals. Furthermore, qPCR-analysis and immunofluorescence on colon muscularis samples revealed that factors associated with neuroinflammation and neurodegeneration, including Bax1, Hdac4, IL-18, Casp8 and Hif1a are overexpressed after DSS-treatment, but not in the case of concurrent l-clodronate administration. Our findings highlight that MM-infiltration in the muscularis layer is responsible for colitis-associated dysmotility and enteric neuronal dysfunction along with the release of mediators associated with neurodegeneration in a murine experimental model.

Abstract Interleukin-1β (IL-1β) is a key mediator of non-alcoholic steatohepatitis (NASH), a chronic liver disease, and of systemic inflammation-driven aging. IL-1β contributes to cardio-metabolic decline, and may promote hepatic oncogenic transformation. Therefore, IL-1β is a potential therapeutic target in these pathologies. We aimed to investigate the hepatic and cardiac effects of an IL-1β targeting monoclonal antibody in an aged mouse model of NASH. 24 months old male C57Bl/6J mice were fed with control or choline deficient (CDAA) diet and were treated with isotype control or anti-IL-1β Mab for 8 weeks. Cardiac functions were assessed by conventional—and 2D speckle tracking echocardiography. Liver samples were analyzed by immunohistochemistry and qRT-PCR. Echocardiography revealed improved cardiac diastolic function in anti-IL-1β treated mice with NASH. Marked hepatic fibrosis developed in CDAA-fed group, but IL-1β inhibition affected fibrosis only at transcriptomic level. Hepatic inflammation was not affected by the IL-1β inhibitor. PCNA staining revealed intensive hepatocyte proliferation in CDAA-fed animals, which was not influenced by neutralization of IL-1β. IL-1β inhibition increased hepatic expression of Pd-1 and Ctla4, while Pd-l1 expression increased in NASH. In conclusion, IL-1β inhibition improved cardiac diastolic function, but did not ameliorate features of NASH; moreover, even promoted hepatic immune checkpoint expression, with concomitant NASH-related hepatocellular proliferation.

Abstract Aberrant Wnt activation has been reported in failing cardiomyocytes. Here we present single cell transcriptome profiling of hearts with inducible cardiomyocyte-specific Wnt activation (β-catΔex3) as well as with compensatory and failing hypertrophic remodeling. We show that functional enrichment analysis points to an involvement of extracellular vesicles (EVs) related processes in hearts of β-catΔex3 mice. A proteomic analysis of in vivo cardiac derived EVs from β-catΔex3 hearts has identified differentially enriched proteins involving 20 S proteasome constitutes, protein quality control (PQC), chaperones and associated cardiac proteins including α-Crystallin B (CRYAB) and sarcomeric components. The hypertrophic model confirms that cardiomyocytes reacted with an acute early transcriptional upregulation of exosome biogenesis processes and chaperones transcripts including CRYAB, which is ameliorated in advanced remodeling. Finally, human induced pluripotent stem cells (iPSC)-derived cardiomyocytes subjected to pharmacological Wnt activation recapitulated the increased expression of exosomal markers, CRYAB accumulation and increased PQC signaling. These findings reveal that secretion of EVs with a proteostasis signature contributes to early patho-physiological adaptation of cardiomyocytes, which may serve as a read-out of disease progression and can be used for monitoring cellular remodeling in vivo with a possible diagnostic and prognostic role in the future.

Abstract Aims Inflammatory pathways are increasingly recognized as an important factor in the pathophysiology of both heart failure (HF) and atrial fibrillation (AF). However, there is no data about inflammation‐related histological and molecular alterations in HF‐associated AF. The objective of our study was to investigate inflammatory pathways and fibrosis in end‐stage HF‐associated AF. Methods and results Left atrial samples of 24 male patients with end stage ischemic HF undergoing heart transplantation were analysed. Twelve patients suffered from sustained AF while the others had no documented AF. The expression of inflammasome sensors and their downstream signalling were investigated by Western blot. No differences were observed in the expression of inflammasome sensors between the two groups, while cleaved caspase‐1 increased tendentiously in the AF group (P = 0.051). Cleaved caspase‐1 also showed significant correlation with the expression of interleukin‐1β and its cleaved form in the total population and in the AF group (P

Little is known about the role of the neuropeptide somatostatin (SST) in myocardial ischemia/reperfusion injury and cardioprotection. Here, we investigated the direct cardiocytoprotective effect of SST on ischemia/reperfusion injury in cardiomyocyte cultures, as well as the expression of SST and its receptors in pig and human heart tissues. SST induced a bell-shaped, concentration-dependent cardiocytoprotection in both adult rat primary cardiomyocytes and H9C2 cells subjected to simulated ischemia/reperfusion injury. Furthermore, in a translational porcine closed-chest acute myocardial infarction model, ischemic preconditioning increased plasma SST-like immunoreactivity. Interestingly, SST expression was detectable at the protein, but not at the mRNA level in the pig left ventricles. SSTR1 and SSTR2, but not the other SST receptors, were detectable at the mRNA level by PCR and sequencing in the pig left ventricle. Moreover, remote ischemic conditioning upregulated SSTR1 mRNA. Similarly, SST expression was also detectable in healthy human interventricular septum samples at the protein level. Furthermore, SST-like immunoreactivity decreased in interventricular septum samples of patients with ischemic cardiomyopathy. SSTR1, SSTR2, and SSTR5 but not SST and the other SST receptors were detectable at the mRNA level by sequencing in healthy human left ventricles. In addition, in healthy human left ventricle samples, SSTR1 and SSTR2 mRNAs were expressed especially in vascular endothelial and some other cell types as detected by RNA Scope®in situ hybridization. This is the first demonstration that SST exerts a direct cardiocytoprotective effect against simulated ischemia/reperfusion injury. Moreover, SST is expressed in the heart tissue at the peptide level; however, it is likely to be of sensory neural origin since its mRNA is not detectable. SSTR1 and SSTR2 might be involved in the cardioprotective action of SST, but other mechanisms cannot be excluded.

BackgroundThe prevalence of non-alcoholic steatohepatitis (NASH) rapidly increases with associated metabolic disorders such as dyslipidemia; therefore, NASH is now considered an independent risk factor of cardiovascular diseases. NASH displays sex-linked epidemiological, phenotypical, and molecular differences; however, little is known about the background of these sex-specific differences on the molecular level.ObjectivesWe aimed to assess sex-specific differences in the expression of inflammatory and fibrotic genes, as well as in cholesterol metabolism, focusing on the expression of Pcsk9 in several tissues in a mouse model of NASH that shows the typical features of the human condition.Methods and ResultsWe fed 10-months-old male and female C57Bl/6J mice with a NASH-inducing CDAA or corresponding control diet for 8 weeks. We found that, compared to the control male mice baseline, hepatic Pcsk9 expression as well as serum PCSK9 level was significantly higher in females, and both circulating PCSK9 level and the hepatic Pcsk9 gene were markedly decreased in female mice during NASH development. Histological analysis revealed that male and female mice develop a similar degree of steatosis; however, fibrosis was more pronounced in males upon CDAA diet feeding. Strikingly, female mice have higher hepatic expression of the pro-inflammatory cytokines (Il1b, Ifng), and increased IL-1β cleavage by the NLRP3 inflammasome, and a decrease in Clec4f+ resident Kupffer cell population in comparison to males in the CDAA-fed groups.ConclusionThis is the first demonstration that there are critical sex-specific differences during NASH development in middle-aged mice regarding inflammation, fibrosis, and cholesterol metabolism and that changes in PCSK9 and IL-1β are likely important contributors to sex-specific changes during the transition to NASH.

Dipeptidyl-peptidase-4 (DPP4) inhibitors are novel medicines for diabetes. The SAVOR-TIMI-53 clinical trial revealed increased heart-failure-associated hospitalization in saxagliptin-treated patients. Although this side effect could limit therapeutic use, the mechanism of this potential cardiotoxicity is unclear. We aimed to establish a cellular platform to investigate DPP4 inhibition and the role of its neuropeptide substrates substance P (SP) and neuropeptide Y (NPY), and to determine the expression of DDP4 and its neuropeptide substrates in the human heart. Western blot, radio-, enzyme-linked immuno-, and RNA scope assays were performed to investigate the expression of DPP4 and its substrates in human hearts. Calcein-based viability measurements and scratch assays were used to test the potential toxicity of DPP4 inhibitors. Cardiac expression of DPP4 and NPY decreased in heart failure patients. In human hearts, DPP4 mRNA is detectable mainly in cardiomyocytes and endothelium. Treatment with DPP4 inhibitors alone/in combination with neuropeptides did not affect viability but in scratch assays neuropeptides decreased, while saxagliptin co-administration increased fibroblast migration in isolated neonatal rat cardiomyocyte-fibroblast co-culture. Decreased DPP4 activity takes part in the pathophysiology of end-stage heart failure. DPP4 compensates against the elevated sympathetic activity and altered neuropeptide tone. Its inhibition decreases this adaptive mechanism, thereby exacerbating myocardial damage.

Extensive evidence from epidemiologic, genetic, and clinical intervention studies has indisputably shown that elevated low-density lipoprotein cholesterol (LDL-C) concentrations play a central role in the pathophysiology of atherosclerotic cardiovascular disease. Apart from LDL-C, also triglycerides independently modulate cardiovascular risk. Reduction of proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a therapeutic target for reducing plasma LDL-C, but it is also associated with a reduction in triglyceride levels potentially through modulation of the expression of free fatty acid transporters. Preclinical data indicate that PCSK9 is up-regulated in the ischaemic heart and decreasing PCSK9 expression impacts on infarct size, post infarct inflammation and remodeling as well as cardiac dysfunction following ischaemia/reperfusion. Clinical data support that notion in that PCSK9 inhibition is associated with reductions in the incidence of myocardial infarction, stroke, and coronary revascularization and an improvement of endothelial function in subjects with increased cardiovascular risk. The aim of the current review is to summarize the current knowledge on the importance of free fatty acid metabolism on myocardial ischaemia/reperfusion injury and to provide an update on recent evidence on the role of hyperlipidemia and PCSK9 in myocardial infarction and cardioprotection.

Abstract Little is known about the molecular mechanism including microRNAs (miRNA) in hypercholesterolemia-induced cardiac dysfunction. We aimed to explore novel hypercholesterolemia-induced pathway alterations in the heart by an unbiased approach based on miRNA omics, target prediction and validation. With miRNA microarray we identified forty-seven upregulated and ten downregulated miRNAs in hypercholesterolemic rat hearts compared to the normocholesterolemic group. Eleven mRNAs with at least 4 interacting upregulated miRNAs were selected by a network theoretical approach, out of which 3 mRNAs (beta-2 adrenergic receptor [Adrb2], calcineurin B type 1 [Ppp3r1] and calcium/calmodulin-dependent serine protein kinase [Cask]) were validated with qRT-PCR and Western blot. In hypercholesterolemic hearts, the expression of Adrb2 mRNA was significantly decreased. ADRB2 and PPP3R1 protein were significantly downregulated in hypercholesterolemic hearts. The direct interaction of Adrb2 with upregulated miRNAs was demonstrated by luciferase reporter assay. Gene ontology analysis revealed that the majority of the predicted mRNA changes may contribute to the hypercholesterolemia-induced cardiac dysfunction. In summary, the present unbiased target prediction approach based on global cardiac miRNA expression profiling revealed for the first time in the literature that both the mRNA and protein product of Adrb2 and PPP3R1 protein are decreased in the hypercholesterolemic heart.

Abstract Background Lipid accumulation in the liver and pancreas is primarily caused by combined hyperlipidemia. However, the effect of isolated hypercholesterolemia without hypertriglyceridemia is not fully described. Therefore, our aim was to investigate whether hypercholesterolemia alone leads to alterations both in hepatic and pancreatic lipid panel and histology in rats. Methods Male Wistar rats were fed with 2% cholesterol +0.25% cholate-supplemented diet or standard chow for 12 weeks. Blood was collected at weeks 0, 4, 8 and 12 to measure serum cholesterol and triglyceride levels. At week 12, both the pancreas and the liver were isolated for further histological and biochemical analysis. Hepatic and plasma fatty acid composition was assessed by gas chromatography. Expression of mRNA of major enzymes involved in saturated/unsaturated fatty acid synthesis was analyzed by qPCR. In separate experiments serum enzyme activities and insulin levels were measured at week 9. Results At week 12, rats fed with 2% cholesterol +0.25% cholate-supplemented diet were characterized by elevated serum cholesterol (4.09 ± 0.20 vs. 2.89 ± 0.22 mmol/L, *p

Abstract Background Cardioprotective value of ischemic post- (IPostC), remote (RIC) conditioning in acute myocardial infarction (AMI) is unclear in clinical trials. To evaluate cardioprotection, most translational animal studies and clinical trials utilize necrotic tissue referred to the area at risk (AAR) by magnetic resonance imaging (MRI). However, determination of AAR by MRI‚ may not be accurate, since MRI-indices of microvascular damage, i.e., myocardial edema and microvascular obstruction (MVO), may be affected by cardioprotection independently from myocardial necrosis. Therefore, we assessed the effect of IPostC, RIC conditioning and ischemic preconditioning (IPreC; positive control) on myocardial necrosis, edema and MVO in a clinically relevant, closed-chest pig model of AMI. Methods and results Acute myocardial infarction was induced by a 90-min balloon occlusion of the left anterior descending coronary artery (LAD) in domestic juvenile female pigs. IPostC (6 × 30 s ischemia/reperfusion after 90-min occlusion) and RIC (4 × 5 min hind limb ischemia/reperfusion during 90-min LAD occlusion) did not reduce myocardial necrosis as assessed by late gadolinium enhancement 3 days after reperfusion and by ex vivo triphenyltetrazolium chloride staining 3 h after reperfusion, however, the positive control, IPreC (3 × 5 min ischemia/reperfusion before 90-min LAD occlusion) did. IPostC and RIC attenuated myocardial edema as measured by cardiac T2-weighted MRI 3 days after reperfusion, however, AAR measured by Evans blue staining was not different among groups, which confirms that myocardial edema is not a measure of AAR, IPostC and IPreC but not RIC decreased MVO. Conclusion We conclude that IPostC and RIC interventions may protect the coronary microvasculature even without reducing myocardial necrosis.

Abstract Background Cell loss and subsequent deterioration of contractile function are hallmarks of chronic heart failure (HF). While apoptosis has been investigated as a participant in the progression of HF, it is unlikely that it accounts for the total amount of non-functional tissue. In addition, there is evidence for the presence of necrotic cardiomyocytes in HF. Therefore, the objective of this study was to investigate the necroptotic proteins regulating necroptosis, a form of programmed necrosis, and thereby assess its potential role in human end-stage HF. Methods Left ventricular samples of healthy controls (C) and patients with end-stage HF due to myocardial infarction (CAD) or dilated cardiomyopathy (DCM) were studied. Immunoblotting for necroptotic and apoptotic markers was performed. Triton X-114 fractionated samples were analyzed to study differences in subcellular localization. Results Elevated expression of RIP1 (receptor-interacting protein), pSer227-RIP3 and its total levels were observed in HF groups compared to controls. On the other hand, caspase-8 expression, a proapoptotic protease negatively regulating necroptosis, was downregulated suggesting activation of necroptosis signaling. Total mixed-lineage kinase domain-like protein (MLKL) expression did not differ among the groups; however, active cytotoxic forms of MLKL were present in all HF samples while they were expressed at almost undetectable levels in controls. Interestingly, pThr357-MLKL unlike pSer358-MLKL, was higher in DCM than CAD. In HF, the subcellular localization of both RIP3 and pThr357-MLKL was consistent with activation of necroptosis signaling. Expression of main apoptotic markers has not indicated importance of apoptosis. Conclusions This is the first evidence showing that human HF of CAD or DCM etiology is positive for markers of necroptosis which may be involved in the development of HF.

(1) Background: Ischemia reperfusion (IR) is the leading cause of acute kidney injury (AKI) and results in predisposition to chronic kidney disease. We demonstrated that delayed contralateral nephrectomy (Nx) greatly improved the function of the IR-injured kidney and decelerated fibrosis progression. Our aim was to identify microRNAs (miRNA/miR) involved in this process. (2) Methods: NMRI mice were subjected to 30 min of renal IR and one week later to Nx/sham surgery. The experiments were conducted for 7–28 days after IR. On day 8, multiplex renal miRNA profiling was performed. Expression of nine miRNAs was determined with qPCR at all time points. Based on the target prediction, plexin-A2 and Cd2AP were measured by Western blot. (3) Results: On day 8 after IR, the expression of 20/1195 miRNAs doubled, and 9/13 selected miRNAs were upregulated at all time points. Nx reduced the expression of several ischemia-induced pro-fibrotic miRNAs (fibromirs), such as miR-142a-duplex, miR-146a-5p, miR-199a-duplex, miR-214-3p and miR-223-3p, in the injured kidneys at various time points. Plexin-A2 was upregulated by IR on day 10, while Cd2AP was unchanged. (4) Conclusion: Nx delayed fibrosis progression and decreased the expression of ischemia-induced fibromirs. The protein expression of plexin-A2 and Cd2AP is mainly regulated by factors other than miRNAs.

Although regular exercise training is associated with cardiovascular benefits, the increased risk of atrial arrhythmias has been observed after vigorous exercise and has been related to oxidative stress. We aimed at investigating exercise-induced atrial remodeling in a rat model of an athlete’s heart and determining sex-specific differences. Age-matched young adult rats were divided into female exercised, female control, male exercised, and male control groups. After exercised animals completed a 12-week-long swim training protocol, echocardiography and in vivo cardiac electrophysiologic investigation were performed. Additionally, atrial histological and gene expression analyses were carried out. Post-mortem atrial weight data and histological examination confirmed marked atrial hypertrophy. We found increased atrial gene expression of antioxidant enzymes along with increased nitro-oxidative stress. No gene expression alteration was found regarding markers of pathological remodeling, apoptotic, proinflammatoric, and profibrotic processes. Exercise training was associated with a prolonged right atrial effective refractory period. We could not induce arrhythmias by programmed stimulation in any groups. We found decreased expression of potassium channels. Female gender was associated with lower profibrotic expression and collagen density. Long-term, balanced exercise training-induced atrial hypertrophy is not associated with harmful electrical remodeling, and no inflammatory or profibrotic response was observed in the atrium of exercised rats.

Cariprazine (Car) is a recently approved second generation antipsychotic (SGA) with unique pharmacodynamic profile, being a partial agonist at both dopamine D2/3 receptor subtypes, with almost 10 times greater affinity towards D3. SGAs are known to increase body weight, alter serum lipids, and stimulate adipogenesis but so far, limited information about the adverse effects is available with this drug. In order to study this new SGA with such a unique mechanism of action, we compared Car to substances that are considered references and are well characterized: olanzapine (Ola) and aripiprazole (Ari). We studied the effects on body weight and also assessed the adipogenesis in rats. The drugs were self-administered in two different doses to female, adult, Wistar rats for six weeks. Weekly body weight change, vacuole size of adipocytes, Sterol Regulatory Element Binding Protein-1 (SREBP-1) and Uncoupling Protein-1 (UCP-1) expression were measured from the visceral adipose tissue (AT). The adipocyte’s vacuole size, and UCP-1 expression were increased while body weight gain was diminished by Car. by increasing UCP-1 might stimulate the thermogenesis, that could potentially explain the weight gain lowering effect through enhanced lipolysis.

Search for new cardioprotective therapies is of great importance since no cardioprotective drugs are available on the market. In line with this need, several natural biomolecules have been extensively tested for their potential cardioprotective effects. Previously, we have shown that biglycan, a member of a diverse group of small leucine-rich proteoglycans, enhanced the expression of cardioprotective genes and decreased ischemia/reperfusion-induced cardiomyocyte death via a TLR-4 dependent mechanism. Therefore, in the present study we aimed to test whether decorin, a small leucine-rich proteoglycan closely related to biglycan, could exert cardiocytoprotection and to reveal possible downstream signaling pathways. Methods: Primary cardiomyocytes isolated from neonatal and adult rat hearts were treated with 0 (Vehicle), 1, 3, 10, 30 and 100 nM decorin as 20 h pretreatment and maintained throughout simulated ischemia and reperfusion (SI/R). In separate experiments, to test the mechanism of decorin-induced cardio protection, 3 nM decorin was applied in combination with inhibitors of known survival pathways, that is, the NOS inhibitor L-NAME, the PKG inhibitor KT-5823 and the TLR-4 inhibitor TAK-242, respectively. mRNA expression changes were measured after SI/R injury. Results: Cell viability of both neonatal and adult cardiomyocytes was significantly decreased due to SI/R injury. Decorin at 1, 3 and 10 nM concentrations significantly increased the survival of both neonatal and adult myocytes after SI/R. At 3nM (the most pronounced protective concentration), it had no effect on apoptotic rate of neonatal cardiac myocytes. No one of the inhibitors of survival pathways (L-NAME, KT-5823, TAK-242) influenced the cardiocytoprotective effect of decorin. MYND-type containing 19 (Zmynd19) and eukaryotic translation initiation factor 4E nuclear import factor 1 (Eif4enif1) were significantly upregulated due to the decorin treatment. In conclusion, this is the first demonstration that decorin exerts a direct cardiocytoprotective effect possibly independent of NO-cGMP-PKG and TLR-4 dependent survival signaling.

The objective of our present study is to develop novel inhibitors for MMP-2 for acute cardioprotection. In a series of pilot studies, novel substituted carboxylic acid derivatives were synthesized based on imidazole and thiazole scaffolds and then tested in a screeening cascade for MMP inhibition. We found that the MMP-inhibiting effects of imidazole and thiazole carboxylic acid-based compounds are superior in efficacy in comparison to the conventional hydroxamic acid derivatives of the same molecules. Based on these results, a 568-membered focused library of imidazole and thiazole compounds was generated in silico and then the library members were docked to the 3D model of MMP-2 followed by an in vitro medium throughput screening (MTS) based on a fluorescent assay employing MMP-2 catalytic domain. Altogether 45 compounds showed a docking score of >70, from which 30 compounds were successfully synthesized. Based on the MMP-2 inhibitory tests using gelatin zymography, 7 compounds were then selected and tested in neonatal rat cardiac myocytes subjected to simulated I/R injury. Six compounds showed significant cardio-cytoprotecion and the most effective compound (MMPI-1154) significantly decreased infarct size when applied at 1 μM in an ex vivo model for acute myocardial infarction. This is the first demonstration that imidazole and thiazole carboxylic acid-based compounds are more efficacious MMP-2 inhibitor than their hydroxamic acid derivatives. MMPI-1154 is a promising novel cardio-cytoprotective imidazole-carboxylic acid MMP-2 inhibitor lead candidate for the treatment of acute myocardial infarction.

Increased oxidative stress is a major contributor to the development and progression of heart failure, however, our knowledge on the role of the distinct NADPH oxidase (NOX) isoenzymes, especially on NOX4 is controversial. Therefore, we aimed to characterize NOX4 expression in human samples from healthy and failing hearts. Explanted human heart samples (left and right ventricular, and septal regions) were obtained from patients suffering from heart failure of ischemic or dilated origin. Control samples were obtained from donor hearts that were not used for transplantation. Deep RNA sequencing of the cardiac transcriptome indicated extensive alternative splicing of the NOX4 gene in heart failure as compared to samples from healthy donor hearts. Long distance PCR analysis with a universal 5′-3′ end primer pair, allowing amplification of different splice variants, confirmed the presence of the splice variants. To assess translation of the alternatively spliced transcripts we determined protein expression of NOX4 by using a specific antibody recognizing a conserved region in all variants. Western blot analysis showed up-regulation of the full-length NOX4 in ischemic cardiomyopathy samples and confirmed presence of shorter isoforms both in control and failing samples with disease-associated expression pattern. We describe here for the first time that NOX4 undergoes extensive alternative splicing in human hearts which gives rise to the expression of different enzyme isoforms. The full length NOX4 is significantly upregulated in ischemic cardiomyopathy suggesting a role for NOX4 in ROS production during heart failure.

Administration of low-dose endotoxin (lipopolysaccharide, LPS) 24 h before a lethal ischemia induces pharmacological late preconditioning. The exact mechanism of this phenomenon is not clear. Here we aimed to investigate whether low-dose LPS exerts late effects on peroxynitrite formation and activation of Akt, Erk, and STAT3 in the heart. Male Wistar rats were injected with LPS (S. typhimurium; 0.5 mg/kg i.p.) or saline. Twenty-four hours later, hearts were isolated, perfused for 10 min, and then used for biochemical analyses. LPS pretreatment enhanced cardiac formation of the peroxynitrite marker 3-nitrotyrosine. LPS pretreatment also increased cardiac levels of the peroxynitrite precursor nitric oxide (NO) and superoxide. The activities of Ca2+-independent NO synthase and xanthine oxidoreductase increased in LPS-pretreated hearts. LPS pretreatment resulted in significantly enhanced phosphorylation of STAT3 and non-significantly increased phosphorylation of Akt without affecting the activation of Erk. In separate experiments, isolated working hearts were subjected to 30 min global ischemia and 20 min reperfusion. LPS pretreatment significantly improved ischemia-reperfusion-induced deterioration of cardiac function. We conclude that LPS pretreatment enhances cardiac peroxynitrite formation and activates STAT3 24 h later, which may contribute to LPS-induced late preconditioning.

Abstract Myocarditis is a major cause of heart failure and sudden cardiac death in young adults and adolescents. Many cases of myocarditis are associated with autoimmune processes in which cardiac myosin is a major autoantigen. Conventional immunosuppressive therapies often provide unsatisfactory results and are associated with adverse toxicities during the treatment of autoimmune myocarditis. Cannabidiol (CBD) is a nonpsychoactive constituent of marijuana that exerts antiinflammatory effects independent of classical cannabinoid receptors. Recently, 80 clinical trials have investigated the effects of CBD in various diseases from inflammatory bowel disease to graft versus host disease. CBD-based formulations are used for the management of multiple sclerosis in numerous countries, and CBD also received U.S. Food and Drug Administration approval for the treatment of refractory childhood epilepsy and glioblastoma multiforme. Herein, using a well-established mouse model of experimental autoimmune myocarditis (EAM) induced by immunization with cardiac myosin emmulsified in adjuvant resulting in T cell-mediated inflammation, cardiomyocyte cell death, fibrosis and myocardial dysfunction, we studied the potential beneficial effects of CBD. EAM was characterized by marked myocardial T-cell infiltration, profound inflammatory response and fibrosis (measured by quantitative real-time polymerase chain reaction, histology and immunohistochemistry analyses) accompanied by marked attenuation of both systolic and diastolic cardiac functions measured with a pressure-volume conductance catheter technique. Chronic treatment with CBD largely attenuated the CD3+ and CD4+ T cell-mediated inflammatory response and injury, myocardial fibrosis and cardiac dysfunction in mice. In conclusion, CBD may represent a promising novel treatment for managing autoimmune myocarditis and possibly other autoimmune disorders and organ transplantation.

Chronic alcohol consumption is a well-known risk factor for tissue injury. The link between alcohol use disorder (AUD) and kidney injury is intriguing but controversial, and the molecular mechanisms by which alcohol may damage the kidneys are poorly understood. Epidemiological studies attempting to link AUD and kidney disease are, to date, inconclusive, and there is little experimental evidence directly linking alcohol consumption to kidney injury. However, studies conducted primarily in other organs and tissues suggest several possible mechanisms by which alcohol may promote kidney dysfunction. One possible mechanism is oxidative stress resulting from increased production of reactive oxygen species, which leads to an excessive amount of free radicals, which in turn trigger tissue injury and increase inflammation. In addition, AUD's effect on other major organs (liver, heart, intestines, and skeletal muscle) appears to promote unfavorable pathological processes that are harmful to the kidneys. Notably, these mechanisms have not yet been validated experimentally in the kidney. Additional research is needed to clarify if alcohol does indeed promote kidney injury and the mechanisms by which alcohol-induced kidney injury may occur.