Synthetic Oligodeoxynucleotide CpG Motifs Activate Human Complement through Their Backbone Structure and Induce Complement-Dependent Cytokine Release.

Bacterial and mitochondrial DNA, sharing an evolutionary origin, act as danger-associated molecular patterns in infectious and sterile inflammation. They both contain immunomodulatory CpG motifs. Interactions between CpG motifs and the complement system are sparsely described, and mechanisms of complement activation by CpG remain unclear. Lepirudin-anticoagulated human whole blood and plasma were incubated with increasing concentrations of three classes of synthetic CpGs: CpG-A, -B, and -C oligodeoxynucleotides and their GpC sequence controls. Complement activation products were analyzed by immunoassays. Cytokine levels were determined via 27-plex beads-based immunoassay, and CpG interactions with individual complement proteins were evaluated using magnetic beads coated with CpG-B. In whole blood and plasma, CpG-B and CpG-C (p < 0.05 for both), but not CpG-A (p > 0.8 for all), led to time- and dose-dependent increase of soluble C5b-9, the alternative complement convertase C3bBbP, and the C3 cleavage product C3bc. GpC-A, -B, and -C changed soluble fluid-phase C5b-9, C3bBbP, and C3bc to the same extent as CpG-A, -B, and -C, indicating a DNA backbone-dependent effect. Dose-dependent CpG-B binding was found to C1q (r = 0.83; p = 0.006) and factor H (r = 0.93; p < 0.001). The stimulatory complement effect was partly preserved in C2-deficient plasma and completely preserved in MASP-2-deficient serum. CpG-B increased levels of IL-1ß, IL-2, IL-6, IL-8, MCP-1, and TNF in whole blood, which were completely abolished by inhibition of C5 and C5aR1 (p < 0.05 for all). In conclusion, synthetic analogs of bacterial and mitochondrial DNA activate the complement system via the DNA backbone. We suggest that CpG-B interacts directly with classical and alternative pathway components, resulting in complement-C5aR1-dependent cytokine release.

When Our Best Friend Becomes Our Worst Enemy: The Mitochondrion in Trauma, Surgery, and Critical Illness.

Common for major surgery, multitrauma, sepsis, and critical illness, is a whole-body inflammation. Tissue injury is able to trigger a generalized inflammatory reaction. Cell death causes release of endogenous structures termed damage associated molecular patterns (DAMPs) that initiate a sterile inflammation. Mitochondria are evolutionary endosymbionts originating from bacteria, containing molecular patterns similar to bacteria. These molecular patterns are termed mitochondrial DAMPs (mDAMPs). Mitochondrial debris released into the extracellular space or into the circulation is immunogenic and damaging secondary to activation of the innate immune system. In the circulation, released mDAMPS are either free or exist in extracellular vesicles, being able to act on every organ and cell in the body. However, the role of mDAMPs in trauma and critical care is not fully clarified. There is a complete lack of knowledge how they may be counteracted in patients. Among mDAMPs are mitochondrial DNA, cardiolipin, N-formyl peptides, cytochrome C, adenosine triphosphate, reactive oxygen species, succinate, and mitochondrial transcription factor A. In this overview, we present the different mDAMPs, their function, release, targets, and inflammatory potential. In light of present knowledge, the role of mDAMPs in the pathophysiology of major surgery and trauma as well as sepsis, and critical care is discussed.

Hyperglycemia-simulating environment attenuated experimentally induced calcification in cultured human aortic valve interstitial cells.

Objectives: The role of diabetes mellitus as a risk factor for the development of calcific aortic valve disease has not been fully clarified. Aortic valve interstitial cells (VICs) have been suggested to be crucial for calcification of the valve. Induced calcification in cultured VICs is a good in vitro model for aortic valve calcification. The purpose of this study was to investigate whether increased glucose levels increase experimentally induced calcification in cultured human VICs. Design: VICs were isolated from explanted calcified aortic valves after valve replacement. Osteogenic medium induced calcification of cultured VICs at different glucose levels (5, 15, and 25 mM). Calcium deposits were visualized using Alizarin Red staining and measured spectrophotometrically. Results: The higher the glucose concentration, the lower the level of calcification. High glucose (25 mM) reduced calcification by 52% compared with calcification at a physiological (5 mM) glucose concentration (correlation and regression analysis: r = -0.55, p = .025 with increased concentration of glucose). Conclusions: In vitro hyperglycemia-like conditions attenuated calcification in VICs. High glucose levels may trigger a series of events that secondarily stimulate calcification of VICs in vivo.

Toll-like receptor 9 signaling after myocardial infarction: Role of p66ShcA adaptor protein.

During myocardial infarction, cellular debris is released, causing a sterile inflammation via pattern recognition receptors. These reactions amplify damage and promotes secondary heart failure. The pattern recognition receptor, Toll-like receptor 9 (TLR9) detects immunogenic fragments of endogenous DNA, inducing inflammation by NFκB. The p66ShcA adaptor protein plays an important role in both ischemic myocardial damage and immune responses. We hypothesized that p66ShcA adaptor protein promotes DNA-sensing signaling via the TLR9 pathway after myocardial infarction. TLR9 protein expression increased in cardiac tissue from patients with end-stage heart failure due to ischemic heart disease. Myocardial ischemia in mice in vivo induced gene expression of key TLR9 pathway proteins (MyD88 and Unc93b1). In this model, a functional link between TLR9 and p66ShcA was revealed as; (i) ischemia-induced upregulation of TLR9 protein was abrogated in myocardium of p66ShcA knockout mice; (ii) when p66ShcA was overexpressed in NFkB reporter cells stably expressing TLR9, NFkB-activation increased during stimulation with the TLR9 agonist CpG B; (iii) in cardiac fibroblasts, p66ShcA overexpression caused TLR9 upregulation. Co-immunoprecipitation showed that ShcA proteins and TLR9 may be found in the same protein complex, which was dissipated upon TLR9 stimulation in vivo. A proximity assay confirmed the co-localization of TLR9 and ShcA proteins. The systemic immune response after myocardial ischemia was dampened in p66ShcA knockout mice as interleukin-4, -17 and -22 expression in mononuclear cells isolated from spleens was reduced. In conclusion, p66ShcA adaptor may be an interaction partner and a regulator of the TLR9 pathway post-infarction.

Plin2 deletion increases cholesteryl ester lipid droplet content and disturbs cholesterol balance in adrenal cortex.

Cholesteryl esters (CEs) are the water-insoluble transport and storage form of cholesterol. Steroidogenic cells primarily store CEs in cytoplasmic lipid droplet (LD) organelles, as contrasted to the majority of mammalian cell types that predominantly store triacylglycerol (TAG) in LDs. The LD-binding Plin2 binds to both CE- and TAG-rich LDs, and although Plin2 is known to regulate degradation of TAG-rich LDs, its role for regulation of CE-rich LDs is unclear. To investigate the role of Plin2 in the regulation of CE-rich LDs, we performed histological and molecular characterization of adrenal glands from Plin2+/+ and Plin2-/- mice. Adrenal glands of Plin2-/- mice had significantly enlarged organ size, increased size and numbers of CE-rich LDs in cortical cells, elevated cellular unesterified cholesterol levels, and increased expression of macrophage markers and genes facilitating reverse cholesterol transport. Despite altered LD storage, mobilization of adrenal LDs and secretion of corticosterone induced by adrenocorticotropic hormone stimulation or starvation were similar in Plin2+/+ and Plin2-/- mice. Plin2-/- adrenals accumulated ceroid-like structures rich in multilamellar bodies in the adrenal cortex-medulla boundary, which increased with age, particularly in females. Finally, Plin2-/- mice displayed unexpectedly high levels of phosphatidylglycerols, which directly paralleled the accumulation of these ceroid-like structures. Our findings demonstrate an important role of Plin2 for regulation of CE-rich LDs and cellular cholesterol balance in the adrenal cortex.

Connective tissue growth factor and bone morphogenetic protein 2 are induced following myocardial ischemia in mice and humans.

We aimed to study the cardiac expression of bone morphogenetic protein 2, its receptor 1 b, and connective tissue growth factor, factors implicated in cardiac embryogenesis, following ischemia/hypoxia, heart failure, and in remodeling hearts from humans and mice. Biopsies from the left ventricle of patients with end-stage heart failure due to dilated cardiomyopathy or coronary artery disease were compared with donor hearts and biopsies from patients with normal heart function undergoing coronary artery bypass grafting. Mouse model of post-infarction remodeling was made by permanent ligation of the left coronary artery. Hearts were analyzed by real-time polymerase chain reaction and Western blotting after 24 hours and after 2 and 4 weeks. Patients with dilated cardiomyopathy and mice post-infarction had increased cardiac expression of connective tissue growth factor. Bone morphogenetic protein 2 was increased in human hearts failing due to coronary artery disease and in mice post-infarction. Gene expression of bone morphogenetic protein receptor 1 beta was reduced in hearts of patients with failure, but increased two weeks following permanent ligation of the left coronary artery in mice. In conclusion, connective tissue growth factor is upregulated in hearts of humans with dilated cardiomyopathy, bone morphogenetic protein 2 is upregulated in remodeling due to myocardial infarction while its receptor 1 b in human failing hearts is downregulated. A potential explanation might be an attempt to engage regenerative processes, which should be addressed by further, mechanistic studies.

Extracellular mtDNA activates NF-κB via toll-like receptor 9 and induces cell death in cardiomyocytes.

Acute myocardial infarction (AMI) causes sterile inflammation, which exacerbates tissue injury. Elevated levels of circulating mitochondrial DNA (mtDNA) have been associated with AMI. We hypothesized that mtDNA triggers an innate immune response via TLR9 and NF-κB activation, causing cardiomyocyte injury. Murine cardiomyocytes express TLR9 mRNA and protein and were able to internalize fluorescently labeled mouse mtDNA. Incubation of human embryonic kidney cells with serum from AMI patients containing naturally elevated levels of mtDNA induced TLR9-dependent NF-κB activity. This effect was mimicked by isolated mtDNA. mtDNA activated NF-κB in reporter mice both in vivo and in isolated cardiomyocytes. Moreover, incubation of isolated cardiomyocytes with mtDNA induced cell death after 4 and 24 h. Laser confocal microscopy showed that incubation of cardiomyocytes with mtDNA accelerated mitochondrial depolarization induced by reactive oxygen species. In contrast to mtDNA, isolated total DNA did not activate NF-κB nor induce cell death. In conclusion, mtDNA can induce TLR9-dependent NF-κB activation in reporter cells and activate NF-κB in cardiomyocytes. In cardiomyocytes, mtDNA causes mitochondrial dysfunction and death. Endogenous mtDNA in the extracellular space is a danger signal with direct detrimental effects on cardiomyocytes.

The p66ShcA adaptor protein regulates healing after myocardial infarction.

Heart rupture and heart failure are deleterious complications of myocardial infarction. The ShcA gene encodes for three protein isoforms, p46-, p52- and p66ShcA. p66ShcA induces oxidative stress. We studied the role of p66ShcA post-infarction. Expression of p66ShcA was analyzed in myocardium of patients with stable angina (n = 11), in explanted hearts with end-stage ischemic heart failure (n = 9) and compared to non-failing hearts not suitable for donation (n = 7). p66ShcA was increased in the patients with stable angina, but not in the patients with end-stage heart failure. Mice (n = 105) were subjected to coronary artery ligation. p66ShcA expression and phosphorylation were evaluated over a 6-week period. p66ShcA expression increased transiently during the first weeks post-infarction. p66ShcA knockout mice (KO) were compared to wild type (n = 82 in total). KO had improved survival and reduced occurrence of heart rupture post-infarction. Expression of cardiac matrix metalloproteinase 2 (MMP-2) was reduced; fibroblast activation and collagen accumulation were facilitated, while oxidative stress was attenuated in KO early post-infarction. 6 weeks post-infarction, reactive fibrosis and left ventricular dilatation were diminished in KO. p66ShcA regulation of MMP-2 was demonstrated in cultured fibroblasts: lack or overexpression of p66ShcA in vitro altered expression of MMP-2. Myocardial infarction induced cardiac p66ShcA. Deletion of p66ShcA improved early survival, myocardial healing and reduced cardiac fibrosis. Upon myocardial infarction p66ShcA regulates MMP-2 activation. The role of p66ShcA in human cardiac disease deserves further study as a potential target for reducing adverse cardiac remodeling post-infarction.

Oxygen- and temperature-dependent expression of survival protein kinases in crucian carp (Carassius carassius) heart and brain.

Living without oxygen is limited to very few vertebrates, one species being the fresh water fish crucian carp (Carassius carassius), which can survive months of anoxia at low temperatures. Mammalian heart and brain are particularly intolerant to oxygen deprivation, yet these organs can be conditioned to display increased resistance, possibly due to activation of several protein kinases. We hypothesized increased phosphorylation status of these kinases in hypoxic and anoxic crucian carp heart and brain. Moreover, we wanted to investigate whether the kinases showing the strongest phosphorylation during hypoxia/anoxia, ERK 1/2, p38-MAPK, JNK, PKCε, and PKCδ, also had increased expression and phosphorylation at cold temperatures, to better cope with the anoxic periods during winter. We found small differences in the phosphorylation status of ERK 1/2, p38-MAPK, JNK, PKCε, and PKCδ during 10 days of severe hypoxia in both heart and brain (0.3 mg O2/l) and varying responses to reoxygenation. In contrast, 7 days of anoxia (<0.01 mg O2/l) markedly increased phosphorylation of ERK 1/2, p38-MAPK, JNK in the heart, and p38-MAPK and PKCε in the brain. Similarly, varying acclimation temperature between 4, 10 and 20°C induced large changes in phosphorylation status. Total protein expression in heart and brain neither changed during different oxygen regimes nor with different acclimation temperatures, except for ERK 1/2, which slightly decreased in the heart at 4°C compared with 20°C. A phylogenetic analysis confirmed that these protein kinases are evolutionarily conserved across a wide range of vertebrate species. Our findings indicate important roles of several protein kinases during oxygen deprivation.

Deletion of the aquaporin-4 gene alters expression and phosphorylation of protective kinases in the mouse heart.

AIM: Aquaporins are channel-forming proteins highly permeable to water and some small molecular solutes. We have previously shown that aquaporin-4 knockout mice have increased tolerance to ischemia. However, the mechanism of cardioprotection was unclear. The aim of the current study was to investigate the effects of aquaporin-4 deletion on baseline expression and phosphorylation of some cardioprotective protein kinases. METHODS: Proteins were extracted from hearts of aquaporin-4 knockout mice and littermate wild-type controls and analyzed with Western blot. Samples were taken from young (≤ 6 months of age), and old (≥ 1 year) mice. RESULTS: Western blots showed three different isoforms of aquaporin-4 in wild types, likely representing M1, M23, and Mz. Total AMP-dependent kinase expression was decreased in aquaporin-4 knockout hearts by 18 ± 13% (p = 0.02), while the expression of Akt kinase, extracellular signal regulated kinase 1/2, protein kinase C-epsilon, mitogen-associated kinase P38 and C-Jun N-terminal kinase was unchanged. The phosphorylation of Akt kinase was reduced in hearts from knockout mice by 41 ± 16% (p = 0.01). No other alterations in phosphorylation were found. These effects were only detected in young mice. CONCLUSION: Deletion of the aquaporin-4 gene decreased AMP-dependent kinase expression and Akt kinase phosphorylation in the heart. These changes may influence energy metabolism and endogenous cardioprotection.

Aquaporin-1 in cardiac endothelial cells is downregulated in ischemia, hypoxia and cardioplegia.

Aquaporin-1 (AQP1) is expressed in human and mouse hearts, but little is known about its cellular and subcellular localization and regulation. The aim of this study was to investigate the localization of AQP1 in the mouse heart and to determine the effects of ischemia and hypoxia on its expression. Mouse myocardial cells were freshly isolated and split into cardiomyocyte and non-cardiomyocyte fractions. Isolated, Langendorff-perfused C57Bl6 mouse hearts (n=46) were harvested with no intervention, subjected to 35min of ischemia or ischemia followed by 60min of reperfusion. Eleven mouse hearts were perfusion-fixed for electron microscopy. Forty C57Bl6 mice were exposed to normobaric hypoxia for one or two weeks (n=12). Needle biopsies of human left ventricular myocardium were sampled (n=30) during coronary artery bypass surgery before cardioplegia and after 30min of reperfusion. Human umbilical vein endothelial cells (HUVECs) were subjected to 4h of hypoxia with reoxygenation for either 4 or 24h. AQP1 expression was studied by electron microscopy with immunogold labeling, Western blot, and qPCR. Expression of miR-214 and miR-320 in HUVECs with hypoxia was studied with qPCR. HUVECs were then transfected with precursors and inhibitors of miR-214. AQP1 expression was confined to cardiac endothelial cells, with no signal in cardiomyocytes or cardiac fibroblasts. Immunogold electron microscopy showed AQP1 expression in endothelial caveolae with equal distribution along the basal and apical membranes. Ischemia and reperfusion tended to decrease AQP1 mRNA expression in mouse hearts by 37±9% (p=0.06), while glycosylated AQP1 protein was reduced by 16±9% (p=0.03). No difference in expression was found between ischemia alone and ischemia-reperfusion. In human left ventricles AQP1 mRNA expression was reduced following cardioplegia and reperfusion (p=0.008). Hypoxia in mice reduced AQP1 mRNA expression by 20±7% (p<0.0001), as well as both glycosylated (-47±10%, p=0.03) and glycan-free protein (-34±16%, p=0.05). Hypoxia and reoxygenation in HUVECs downregulated glycan-free AQP1 protein (-34±24%, p=0.04) and upregulated miR-214 (+287±52%, p<0.05). HUVECs transfected with anti-miR-214 had increased glycosylated (1.5 fold) and glycan-free (2 fold) AQP1. AQP1 in mouse hearts is localized to endothelial cell membranes and caveolae. Cardioplegia, ischemia and hypoxia decrease AQP1 mRNA as well as total protein expression and glycosylation, possibly regulated by miR-214.

Cardiac aquaporins.

Aquaporins are a group of proteins with high-selective permeability for water. A subgroup called aquaglyceroporins is also permeable to glycerol, urea and a few other solutes. Aquaporin function has mainly been studied in the brain, kidney, glands and skeletal muscle, while the information about aquaporins in the heart is still scarce. The current review explores the recent advances in this field, bringing aquaporins into focus in the context of myocardial ischemia, reperfusion, and blood osmolarity disturbances. Since the amount of data on aquaporins in the heart is still limited, examples and comparisons from better-studied areas of aquaporin biology have been used. The human heart expresses aquaporin-1, -3, -4 and -7 at the protein level. The potential roles of aquaporins in the heart are discussed, and some general phenomena that the myocardial aquaporins share with aquaporins in other organs are elaborated. Cardiac aquaporin-1 is mostly distributed in the microvasculature. Its main role is transcellular water flux across the endothelial membranes. Aquaporin-4 is expressed in myocytes, both in cardiac and in skeletal muscle. In addition to water flux, its function is connected to the calcium signaling machinery. It may play a role in ischemia-reperfusion injury. Aquaglyceroporins, especially aquaporin-7, may serve as a novel pathway for nutrient delivery into the heart. They also mediate toxicity of various poisons. Aquaporins cannot influence permeability by gating, therefore, their function is regulated by changes of expression-on the levels of transcription, translation (by microRNAs), post-translational modification, membrane trafficking, ubiquitination and subsequent degradation. Studies using mice genetically deficient for aquaporins have shown rather modest changes in the heart. However, they might still prove to be attractive targets for therapy directed to reduce myocardial edema and injury caused by ischemia and reperfusion.

Mitochondria-derived damage-associated molecular patterns and inflammation in the ischemic-reperfused heart.

Cardiac cell death after myocardial infarction release endogenous structures termed damage-associated molecular patterns (DAMPs) that trigger the innate immune system and initiate a sterile inflammation in the myocardium. Cardiomyocytes are energy demanding cells and 30% of their volume are mitochondria. Mitochondria are evolutionary endosymbionts originating from bacteria containing molecular patterns similar to bacteria, termed mitochondrial DAMPs (mDAMPs). Consequently, mitochondrial debris may be particularly immunogenic and damaging. However, the role of mDAMPs in myocardial infarction is not clarified. Identifying the most harmful mDAMPs and inhibiting their early inflammatory signaling may reduce infarct size and the risk of developing post-infarct heart failure. The focus of this review is the role of mDAMPs in the immediate pro-inflammatory phase after myocardial infarction before arrival of immune cells in the myocardium. We discuss different mDAMPs, their role in physiology and present knowledge regarding their role in the inflammatory response of acute myocardial infarction.

Escherichia coli-induced inflammatory responses are temperature-dependent in human whole blood ex vivo.

Systemic inflammatory conditions are often associated with hypothermia or hyperthermia. Therapeutic hypothermia is used in post-cardiac arrest and some other acute diseases. There is a need for more knowledge concerning the effect of various temperatures on the acute inflammatory response. The complement system plays a crucial role in initiating the inflammatory response. We hypothesized that temperatures above and below the physiologic 37 °C affect complement activation and cytokine production ex vivo. Lepirudin-anticoagulated human whole blood from 10 healthy donors was incubated in the presence or absence of Escherichia coli at different temperatures (4 °C, 12 °C, 20 °C, 33 °C, 37 °C, 39 °C, and 41 °C). Complement activation was assessed by the terminal C5b-9 complement complex (TCC) and the alternative convertase C3bBbP using ELISA. Cytokines were measured using a 27-plex assay. Granulocyte and monocyte activation was evaluated by CD11b surface expression using flow cytometry. A consistent increase in complement activation was observed with rising temperature, reaching a maximum at 41 °C, both in the absence (C3bBbP p < 0.05) and presence (C3bBbP p < 0.05 and TCC p < 0.05) of E. coli. Temperature alone did not affect cytokine production, whereas incubation with E. coli significantly increased cytokine levels of IL-1ß, IL-2, IL-6, IL-8, IFN-γ, and TNF at temperatures > 20 °C. Maximum increase occurred at 39 °C. However, a consistent decrease was observed at 41 °C, significant for IL-1ß (p = 0.003). Granulocyte CD11b displayed the same temperature-dependent pattern as cytokines, with a corresponding increase in endothelial cell apoptosis and necrosis. Thus, blood temperature differentially determines the degree of complement activation and cytokine release.

Transient hyperosmolality modulates expression of cardiac aquaporins.

PURPOSE: Hyperosmolarity is a common complication in intensive care patients, dysregulating water balance in many organs including brain and heart. The aquaporin (AQP) water channels, in particular AQP1 and -4, have been suggested to play an important role in fluid homeostasis of the myocardium. In many organs AQP expression is regulated by osmolarity, drastically altering water permeability of the cell membranes. The aim of our study was to investigate if plasma hyperosmolality may regulate cardiac expression of AQP1 and -4, and if so, at which magnitude and time frame such regulation takes place. METHODS: C57Bl6 mice were injected intraperitoneally with either 1.5 ml 0.154 Mol (isoosmotic), 0.5 ml 1 Mol (mild hyperosmotic) or 0.5 ml 2 Mol (strong hyperosmotic) NaCl. Plasma, hearts, and forebrains were harvested before injection ("time 0"), and after 1, 4, 8 and 24 h. AQP1 and -4 expression were analyzed using qPCR and Western blot. RESULTS: Isoosmotic and mild hyperosmotic injections caused no important changes in cardiac AQP expression. Strong hyperosmotic NaCl injections induced an upregulation of AQP1 mRNA and glycosylated fraction of AQP1 protein in the heart without changes of the total protein. AQP4 mRNA and protein decreased in the heart and increased in the brain after hyperosmotic NaCl. The change in AQP4 protein content in the brain preceded the increase of mRNA. CONCLUSION: As in the brain, expression of AQP1 and -4 in the heart is influenced by changes in plasma osmolality. Changes in AQP expression may alter cardiac function in hyperosmotic states.

Aquaporin-4 in the heart: expression, regulation and functional role in ischemia.

Aquaporins (AQPs) are channel-forming membrane proteins highly permeable to water. AQP4 is found in mammalian hearts; however, its expression sites, regulation and function are largely unknown. The aim was to investigate cardiac AQP4 expression in humans and mice, its regulation by ischemia and hypoxia, and in particular its role in cardiac ischemic injury using AQP4 knockout (KO) mice. Comparable levels of AQP4 were detected by Western blot and qPCR in biopsies from human donor hearts and wild type C57Bl6 mouse hearts. In mice, AQP4 was expressed on cardiomyocyte plasmalemma (qPCR, Western blot, immunogold), and its mRNA decreased following ischemia/reperfusion (isolated hearts, p = 0.02) and after normobaric hypoxia in vivo (oxygen fraction 10 % for 1 week, p < 0.001). Isolated hearts from AQP4 KO mice undergoing global ischemia and reperfusion had reduced infarct size (p = 0.05) and attenuated left ventricular end-diastolic pressure during reperfusion (p = 0.04). Infarct size was also reduced in AQP4 KO mice 24 h after left coronary artery ligation in vivo (p = 0.036). AQP4 KO hearts had no compensatory change in AQP1 protein expression. AQP4 KO cardiomyocytes were partially resisted to hypoosmotic stress in the presence of hypercontracture. AQP4 is expressed in human and mouse hearts, in the latter confined to the cardiomyocyte plasmalemma. AQP4 mRNA expression is downregulated by hypoxia and ischemia. Deletion of AQP4 is protective in acute myocardial ischemia-reperfusion, and this molecule might be a future target in the treatment of acute myocardial infarction.

Effects of ischaemic postconditioning in aortic valve replacement: a multicenter randomized controlled trial.

OBJECTIVES: The effect of ischaemic postconditioning (IPost) on postcardioplegic cardiac function is not known. We hypothesized that IPost was cardioprotective in adult patients undergoing elective aortic valve replacement. METHODS: In a multicentre, prospective, randomized trial, patients (n = 209) were randomized to either a standard operation (controls) or postconditioning. Immediately before the cross-clamp was released, patients in the postconditioning group underwent 3 cycles of flow/non-flow (2 min each) of normothermic blood via the antegrade cardioplegia line. The primary end point was cardiac index. Secondary end points included additional haemodynamic measurements, biomarkers of cardiomyocyte injury, renal function parameters, intra- and postoperative arrhythmias and use of inotropic agents. RESULTS: There was no significant difference between the groups regarding cardiac index [mean between-group difference, 95% confidence interval (CI), 0.11 (-0.1 to 0.3), P = 0.27]. Postconditioning had no effect on other haemodynamic parametres. There was no between-group difference regarding troponin T or creatine kinase MB. Postconditioning reduced the relative risk for arrhythmias by 45% (P = 0.03) when postoperative atrial fibrillation and intraoperative ventricular fibrillation were combined. There were no differences in patients with/without diabetes, patients above/below 70 years of age or between the centres. However, after postconditioning, the cardiac index [95% CI, 0.46 (0.2-0.7), P = 0.001], cardiac output (P < 0.001), mean arterial pressure (P < 0.001) and left ventricular stroke work index (P < 0.001) were higher in males compared to females. CONCLUSIONS: IPost had no overall cardioprotective effects in patients undergoing aortic valve replacement but improved postoperative cardiac performance in men compared to women.

Multi-omics of in vitro aortic valve calcification.

Heart valve calcification is an active cellular and molecular process that partly remains unknown. Osteogenic differentiation of valve interstitial cells (VIC) is a central mechanism in calcific aortic valve disease (CAVD). Studying mechanisms in CAVD progression is clearly needed. In this study, we compared molecular mechanisms of osteogenic differentiation of human VIC isolated from healthy donors or patients with CAVD by RNA-seq transcriptomics in early timepoint (48 h) and by shotgun proteomics at later timepoint (10th day). Bioinformatic analysis revealed genes and pathways involved in the regulation of VIC osteogenic differentiation. We found a high amount of stage-specific differentially expressed genes and good accordance between transcriptomic and proteomic data. Functional annotation of differentially expressed proteins revealed that osteogenic differentiation of VIC involved many signaling cascades such as: PI3K-Akt, MAPK, Ras, TNF signaling pathways. Wnt, FoxO, and HIF-1 signaling pathways were modulated only at the early timepoint and thus probably involved in the commitment of VIC to osteogenic differentiation. We also observed a significant shift of some metabolic pathways in the early stage of VIC osteogenic differentiation. Lentiviral overexpression of one of the most upregulated genes (ZBTB16, PLZF) increased calcification of VIC after osteogenic stimulation. Analysis with qPCR and shotgun proteomics suggested a proosteogenic role of ZBTB16 in the early stages of osteogenic differentiation.

Models and Techniques to Study Aortic Valve Calcification in Vitro, ex Vivo and in Vivo. An Overview.

Aortic valve stenosis secondary to aortic valve calcification is the most common valve disease in the Western world. Calcification is a result of pathological proliferation and osteogenic differentiation of resident valve interstitial cells. To develop non-surgical treatments, the molecular and cellular mechanisms of pathological calcification must be revealed. In the current overview, we present methods for evaluation of calcification in different ex vivo, in vitro and in vivo situations including imaging in patients. The latter include echocardiography, scanning with computed tomography and magnetic resonance imaging. Particular emphasis is on translational studies of calcific aortic valve stenosis with a special focus on cell culture using human primary cell cultures. Such models are widely used and suitable for screening of drugs against calcification. Animal models are presented, but there is no animal model that faithfully mimics human calcific aortic valve disease. A model of experimentally induced calcification in whole porcine aortic valve leaflets ex vivo is also included. Finally, miscellaneous methods and aspects of aortic valve calcification, such as, for instance, biomarkers are presented.

Isolated Plin5-deficient cardiomyocytes store less lipid droplets than normal, but without increased sensitivity to hypoxia.

Plin5 is abundantly expressed in the heart where it binds to lipid droplets (LDs) and facilitates physical interaction between LDs and mitochondria. We isolated cardiomyocytes from adult Plin5+/+ and Plin5-/- mice to study the role of Plin5 for fatty acid uptake, LD accumulation, fatty acid oxidation, and tolerance to hypoxia. Cardiomyocytes isolated from Plin5-/- mice cultured with oleic acid stored less LDs than Plin5+/+, but comparable levels to Plin5+/+ cardiomyocytes when adipose triglyceride lipase activity was inhibited. The ability to oxidize fatty acids into CO2 was similar between Plin5+/+ and Plin5-/- cardiomyocytes, but Plin5-/- cardiomyocytes had a transient increase in intracellular fatty acid oxidation intermediates. After pre-incubation with oleic acids, Plin5-/- cardiomyocytes retained a higher content of glycogen and showed improved tolerance to hypoxia compared to Plin5+/+. In isolated, perfused hearts, deletion of Plin5 had no important effect on ventricular pressures or infarct size after ischemia. Old Plin5-/- mice had reduced levels of cardiac triacylglycerides, increased heart weight, and apart from modest elevated expression of mRNAs for beta myosin heavy chain Myh7 and the fatty acid transporter Cd36, other genes involved in fatty acid oxidation, glycogen metabolism and glucose utilization were essentially unchanged by removal of Plin5. Plin5 seems to facilitate cardiac LD storage primarily by repressing adipose triglyceride lipase activity without altering cardiac fatty acid oxidation capacity. Expression of Plin5 and cardiac LD content of isolated cardiomyocytes has little importance for tolerance to acute hypoxia and ischemia, which contrasts the protective role for Plin5 in mouse models during myocardial ischemia.