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.

Inhibiting nucleolin reduces inflammation induced by mitochondrial DNA in cardiomyocytes exposed to hypoxia and reoxygenation.

BACKGROUND AND PURPOSE: Cellular debris causes sterile inflammation after myocardial infarction. Mitochondria constitute about 30 percent of the human heart. Mitochondrial DNA (mtDNA) is a damage-associated-molecular-pattern that induce injurious sterile inflammation. Little is known about mtDNA's inflammatory signalling pathways in cardiomyocytes and how mtDNA is internalized to associate with its putative receptor, toll-like receptor 9 (TLR9). EXPERIMENTAL APPROACH: We hypothesized that mtDNA can be internalized in cardiomyocytes and induce an inflammatory response. Adult mouse cardiomyocytes were exposed to hypoxia-reoxygenation and extracellular DNA. Microscale thermophoresis was used to demonstrate binding between nucleolin and DNA. KEY RESULTS: Expression of the pro-inflammatory cytokines IL-1ß and TNFα were upregulated by mtDNA, but not by nuclear DNA (nDNA), in cardiomyocytes exposed to hypoxia-reoxygenation. Blocking the RNA/DNA binding protein nucleolin with midkine reduced expression of IL-1ß/TNFα and the nucleolin inhibitor AS1411 reduced interleukin-6 release in adult mouse cardiomyocytes. mtDNA bound 10-fold stronger than nDNA to nucleolin. In HEK293-NF-κB reporter cells, mtDNA induced NF-κB activity in normoxia, while CpG-DNA and hypoxia-reoxygenation, synergistically induced TLR9-dependent NF-κB activity. Protein expression of nucleolin was found in the plasma membrane of cardiomyocytes and inhibition of nucleolin with midkine inhibited cellular uptake of CpG-DNA. Inhibition of endocytosis did not reduce CpG-DNA uptake in cardiomyocytes. CONCLUSION AND IMPLICATIONS: mtDNA, but not nDNA, induce an inflammatory response in mouse cardiomyocytes during hypoxia-reoxygenation. In cardiomyocytes, nucleolin is expressed on the membrane and blocking nucleolin reduce inflammation. Nucleolin might be a therapeutic target to prevent uptake of immunogenic DNA and reduce inflammation. LINKED ARTICLES: This article is part of a themed section on Mitochondrial Pharmacology: Featured Mechanisms and Approaches for Therapy Translation. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.22/issuetoc.

Mechanical stress alters the expression of calcification-related genes in vascular interstitial and endothelial cells.

OBJECTIVES: Vascular wall calcification is a major pathophysiological component of atherosclerotic disease with many similarities to osteogenesis. Mechanical stress of the vascular wall may theoretically contribute to the proliferative processes by endothelial and interstitial cells. The aim of the study was to investigate the effect of mechanical stress on the expression of some calcification-related genes in primary human endothelial and interstitial cells, and how endothelial cells may stimulate the fibroblast and smooth muscle cells. METHODS: Human umbilical vein endothelial and interstitial cells were subjected to cyclic stretch using a FlexCell® bioreactor, and interstitial cells were also subjected to tensile strain in cultures embedded in 3-dimensional collagen gels. The medium from endothelial cells was used to stimulate the gel-cultured interstitial cells, or the endothelium was sown directly on top. For comparison, human endothelial and smooth muscle cells were isolated from aortic wall fragments of patients with and without the aortic aneurysm. The expression of genes was measured using quantitative PCR. RESULTS: Four hours of cyclic stretch applied to cultured endothelial cells upregulated the mRNA expression of bone morphogenetic protein 2 (BMP-2), a major procalcific growth factor. When applied to a 3-dimensional culture of vascular interstitial cells, the medium from prestretched endothelial cells decreased the expression of BMP-2 and periostin mRNA in the fibroblasts. The static tension in gel-cultured interstitial cells upregulated BMP-2 mRNA expression. The addition of endothelial cells on the top of this culture also reduced mRNA of anticalcific genes, periostin and osteopontin. Similar changes were observed in smooth muscle cells from human aortic aneurysms compared to cells from the healthy aorta. Aortic aneurysm endothelial cells also showed an increased expression of BMP-2 mRNA. CONCLUSIONS: Endothelial cells respond to mechanical stress by upregulation of pro-osteogenic factor BMP-2 mRNA and modulate the expression of other osteogenic factors in vascular interstitial cells. Endothelial cells may, thus, contribute to vascular calcification when exposed to mechanical stress.

Release of Mitochondrial and Nuclear DNA During On-Pump Heart Surgery: Kinetics and Relation to Extracellular Vesicles.

During heart surgery with cardiopulmonary bypass (CPB), the release of mitochondrial (mtDNA) and nuclear DNA (nDNA) and their association to extracellular vesicles were investigated. In patients undergoing elective coronary artery bypass grafting (CABG, n = 12), blood was sampled before, during, and after surgery from peripheral artery, pulmonary artery, and the coronary sinus. Plasma was separated in three fractions: microvesicles, exosomes, and supernatant. mtDNA and nDNA were measured by qPCR. mtDNA and nDNA levels increased after start of surgery, but before CPB, and increased further during CPB. mtDNA copy number was about 1000-fold higher than nDNA. mtDNA was predominantly localized to the vesicular fractions in plasma, whereas nDNA was predominantly in the supernatant. The amount of free mtDNA increased after surgery. There was no net release or disappearance of DNAs across the pulmonary, systemic, or coronary circulation. Extracellular DNAs, in particular mtDNA, may be important contributors to the whole-body inflammation during CPB.

A dose-response study of glutamate supplementation in isolated, perfused rat hearts undergoing ischaemia and cold cardioplegia.

OBJECTIVES: Several studies have reported superior post-cardioplegic recovery after glutamate supplementation. The optimum dose of glutamate supplementation is unknown. The purpose of this study was to find the optimal protective concentration of glutamate supplementation in a model of ischaemia/cardioplegia and reperfusion. METHODS: Isolated rat hearts (n = 77) were perfused with the Krebs-Henseleit buffer. After stabilization, the hearts were subjected to 25 min of normothermic ischaemia followed by a single 3-min infusion of cold (4-6 °C) St. Thomas' Hospital II cardioplegia and 87 min of cardioplegic ischaemic arrest and 60 min of reperfusion. Sodium-l-glutamate was added to the perfusate (control group had zero glutamate) in increasing concentrations (0.01, 0.1, 1, 10, 20, 30 and 100 mM) and given throughout perfusion. Corresponding concentrations were added to the cardioplegic solution. A balloon in the left ventricle inserted via the left atrium measured left ventricular pressures isometrically. Left ventricular developed pressure was calculated. Myocardial exchange of glucose and lactate was measured prior to ischaemia and during reperfusion. Myocardial content of glycogen and glutamate was measured at the end of reperfusion. RESULTS: During reperfusion left ventricular developed pressure increased (P < 0.0001) in groups supplemented with 0.1, 1.0, 10, 20 and 30 mM glutamate, whereas left ventricular end-diastolic pressure was attenuated (P = 0.008) when compared with the controls. No additional benefit on the continuous data left ventricular developed pressure and left ventricular end-diastolic pressure was observed with glutamate concentrations above 1 mM. Onset of LV pressure rise during the period of ischaemia was delayed by 100 mM of glutamate (P = 0.02). Myocardial content of glutamate was increased in a dose-related manner in Groups 10, 20, 30 and 100 compared with the control hearts (P < 0.0001). Glycogen was increased in the hearts supplemented with 100 mM of glutamate (P = 0.02). CONCLUSIONS: Even low concentrations of l-glutamate improved postischaemic and post-cardioplegic heart function and 1 mM seems to be optimal.

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.

Higher TNFα responses in young males compared to females are associated with attenuation of monocyte adenylyl cyclase expression.

Tumor necrosis factor α (TNFα) expression is strongly attenuated by the intracellular signaling mediator cyclic adenosine monophosphate (cAMP), which is synthesized by adenylyl cyclase (AC) enzymes. We have compared AC regulation and TNFα production in male and female monocytes, and characterized the role of monocyte AC isoforms in TNFα regulation. Males and females, age groups 20-30 years and 50-70 years donated blood for this study. In lipopolysaccharide-stimulated blood from young male donors, we observed significantly higher TNFα responses (6h, p=0.03) compared to females of the same age, a difference not observed in the older donors. Rapid down-regulation of the monocyte AC isoforms AC4, AC7 and AC9 were observed in young males. AC-directed siRNA experiments in the human monocyte cell line THP-1 demonstrated that AC7 and AC9 knock-down significantly induced TNFα release (p=0.01 for both isoforms). These data indicate that the stronger TNFα-responses in young males may be partly associated with male-specific down-regulation of adenylyl cyclases.

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.

Heme oxygenase-1: an emerging therapeutic target to curb cardiac pathology.

Activation of heme oxygenase-1 (HO-1), a heme-degrading enzyme responsive to a wide range of cellular stress, is traditionally considered to convey adaptive responses to oxidative stress, inflammation and vasoconstriction. These diversified effects are achieved through the degradation of heme to carbon monoxide (CO), biliverdin (which is rapidly converted to bilirubin by biliverdin reductase) and ferric iron. Recent findings have added antiproliferative and angiogenic effects to the list of HO-1/CO actions. HO-1 along with its reaction products bilirubin and CO are protective against ischemia-induced injury (myocardial infarction, ischemia-reperfusion (IR)-injury and post-infarct structural remodelling). Moreover, HO-1, and CO in particular, possess acute antihypertensive effects. As opposed to these curative potentials, the long-believed protective effect of HO-1 in cardiac remodelling in response to pressure overload and type 2 diabetes mellitus (DM) has been questioned by recent work. These challenges, coupled with emerging regulatory mechanisms, motivate further in-depth studies to help understand untapped layers of HO-1 regulation and action. The outcomes of these efforts may shed new light on critical mechanisms that could be used to harness the protective potential of this enzyme for the therapeutic benefit of patients suffering from such highly prevalent cardiovascular disorders.

The cellular prion protein counteracts cardiac oxidative stress.

AIMS: The cellular prion protein, PrP(C), whose aberrant isoforms are related to prion diseases of humans and animals, has a still obscure physiological function. Having observed an increased expression of PrP(C) in two in vivo paradigms of heart remodelling, we focused on isolated mouse hearts to ascertain the capacity of PrP(C) to antagonize oxidative damage induced by ischaemic and non-ischaemic protocols. METHODS AND RESULTS: Hearts isolated from mice expressing PrP(C) in variable amounts were subjected to different and complementary oxidative perfusion protocols. Accumulation of reactive oxygen species, oxidation of myofibrillar proteins, and cell death were evaluated. We found that overexpressed PrP(C) reduced oxidative stress and cell death caused by post-ischaemic reperfusion. Conversely, deletion of PrP(C) increased oxidative stress during both ischaemic preconditioning and perfusion (15 min) with H2O2. Supporting its relation with intracellular systems involved in oxidative stress, PrP(C) was found to influence the activity of catalase and, for the first time, the expression of p66(Shc), a protein implicated in oxidative stress-mediated cell death. CONCLUSIONS: Our data demonstrate that PrP(C) contributes to the cardiac mechanisms antagonizing oxidative insults.

Remote transplantation of mesenchymal stem cells protects the heart against ischemia-reperfusion injury.

Cardioprotection can be evoked through extracardiac approaches. This prompted us to investigate whether remote transplantation of stem cells confers protection of the heart against ischemic injury. The cardioprotective effect of subcutaneous transplantation of naïve versus heme oxygenase-1 (HMOX-1)-overexpressing mouse mesenchymal stem cells (MSC) to mice was investigated in hearts subjected to ischemia-reperfusion in a Langendorff perfusion system. Mice were transplanted into the interscapular region with naïve or HMOX-1 transfected MSC isolated from transgenic luciferase reporter mice and compared to sham-treated animals. The fate of transplanted cells was followed by in vivo bioluminescence imaging, revealing that MSC proliferated, but did not migrate detectably from the injection site. Ex vivo analysis of the hearts showed that remote transplantation of mouse adipose-derived MSC (mASC) resulted in smaller infarcts and improved cardiac function after ischemia-reperfusion compared to sham-treated mice. Although HMOX-1 overexpression conferred cytoprotective effects on mASC against oxidative stress in vitro, no additive beneficial effect of HMOX-1 transfection was noted on the ischemic heart. Subcutaneous transplantation of MSC also improved left ventricular function when transplanted in vivo after myocardial infarction. Plasma analysis and gene expression profile of naïve- and HMOX-1-mASC after transplantation pointed toward pentraxin 3 as a possible factor involved in the remote cardioprotective effect of mASC. These results have significant implications for understanding the behavior of stem cells after transplantation and development of safe and noninvasive cellular therapies with clinical applications. Remote transplantation of MSC can be considered as an alternative procedure to induce cardioprotection.

Expression of bone morphogenetic protein 4 and its receptors in the remodeling heart.

AIMS: Heart failure is associated with activation of fetal gene programs. Bone morphogenetic proteins (BMPs) regulate embryonic development through interaction with BMP receptors (BMPRs) on the cell surface. We investigated if the expression of BMP4 and its receptors BMPR1a and BMPR2 were activated in post-infarction remodeling and heart failure. MAIN METHODS: Left ventricular biopsies were taken from explanted hearts of patients with end-stage heart failure due to dilated cardiomyopathy (CMP; n=15) or ischemic heart disease (CAD; n=9), and compared with homograft control preparations from organ donors deceased due to non-cardiac causes (n=7). Other samples were taken from patients undergoing coronary artery bypass grafting (CABG; n=11). Mice were subjected to induced infarction by permanent coronary artery ligation or sham operation, and hearts were sampled serially thereafter (n=7 at each time point). KEY FINDINGS: Human and mouse hearts expressed BMP4 and both receptor subtypes. CABG and CMP patients had increased expression of mRNA encoding for BMP4, but unchanged protein. Mouse hearts had increased BMP4 precursor protein 24h after infarction. BMPR1a protein decreased in CAD patients and initially in postinfarcted mouse hearts, but increased again in the latter after two weeks. Human recombinant BMP4 promoted survival after H2O2 injury in HL-1 cells, and also protected adult mouse cardiomyocytes against hypoxia-reoxygenation injury. SIGNIFICANCE: Adult hearts express BMP4, the mRNA increasingly so in patients with coronary artery disease with good cardiac function. BMPRs are downregulated in cardiac remodeling and failure. Recombinant BMP4 has protective effects on cultured cardiomyocytes.

Expression of retinoic acid target genes in coronary artery disease.

Coronary atherosclerosis can lead to myocardial infarction, and secondarily to post-infarct remodelling and heart failure. Retinoic acid (RA) influences cell proliferation. We hypothesized that RA could influence gene expression and proliferation of cardiovascular cells. Left ventricular biopsies from patients with end-stage heart failure due to coronary artery disease (CAD) or dilated cardiomyopathy were investigated for the content of RA metabolites using liquid chromatography mass spectrometry (LC-MS/MS), and compared with healthy donors. All-trans retinoic acid (ATRA) was increased in the hearts of CAD patients. Gene expression (quantitative PCR) of RA target genes was not influenced in failing hearts, but was increased in the hearts of patients with CAD undergoing open heart surgery. The expression of RA target genes was increased in atherosclerotic lesions from carotid arteries compared to healthy arteries. Stimulation of cardiomyocytes, cardiofibroblasts, smooth muscle cells and endothelial cells with ATRA increased the gene expression of the key enzymes. Cardiofibroblast and smooth muscle cell proliferation were reduced by ATRA, which increased endothelial cell proliferation. Coronary artery disease leads to increased expression of RA target genes. ATRA accumulated in the failing human heart. All investigated cell types present in the heart had induced expression of RA target genes when stimulated with ATRA, which also influenced cell proliferation.

A novel glutamate transporter blocker, LL-TBOA, attenuates ischaemic injury in the isolated, perfused rat heart despite low transporter levels.

OBJECTIVES: Loss of glutamate from cardiomyocytes during ischaemia may aggravate ischaemia-reperfusion injury in open heart surgery. This may be due to reversal of excitatory amino acid transporters (EAATs). However, the expression of such transporters in cardiomyocytes is ambiguous and quantitative data are lacking. Our objective was to study whether EAATs were expressed in the rat heart and to study whether blocking of transporter operation during cardiac ischaemia could be beneficial. METHODS: We used TaqMan real-time PCR and immunoisolation followed by western blotting to unequivocally identify EAAT subtypes in rat hearts. We used a novel high-affinity non-transportable competitive inhibitor, named LL-TBOA [(2S,3S)-3-(3-(6-(6-(2-(2-(2-(2-(2-aminoethoxy)ethoxy)-ethoxy)ethoxy) acetamido)hexanamido)- hexanamido)-5-(4-(trifluoromethyl)benzamido)benzyloxy) aspartic acid], to block EAAT-mediated transport during global ischaemia and reperfusion of isolated rat hearts. RESULTS: Rat hearts expressed EAAT subtypes 1 and 3, while subtypes 2 and 4 were not detected. Hearts were isolated and perfused with 1.6 µM LL-TBOA for 5 min before 30 min of induced global ischaemia and 60 min of reperfusion (n = 8). Control hearts were perfused either with the solvent dimethylsulfoxide 3.5 mM (n = 7) or with no pretreatment (n = 8). Infarct size was evaluated by triphenyl tetrazolium chloride (TTC) staining. LL-TBOA reduced infarct size from 33 ± 14 to 20 ± 5% (mean ± SD) (P = 0.015). Dimethylsulfoxide alone had no effect (35 ± 2%). Reperfusion arrhythmias were reduced by LL-TBOA (P = 0.009), but not by dimethylsulfoxide alone. CONCLUSION: Rat hearts express EAAT1 and EAAT3, but the mRNA levels are, respectively, ∼ 25 and 200 times lower than in the brain. Addition of LL-TBOA has a beneficial effect against ischaemia-reperfusion injury.

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.

Connective tissue growth factor/CCN2 attenuates ß-adrenergic receptor responsiveness and cardiotoxicity by induction of G protein-coupled receptor kinase-5 in cardiomyocytes.

Myocardial connective tissue growth factor (CTGF/CCN2) is induced in heart failure, a condition associated with diminution of ß-adrenergic receptor (ß-AR) responsiveness. Accordingly, we aimed to investigate whether CTGF could play a mechanistic role in regulation of ß-AR responsiveness. Concentration-response curves of isoproterenol-stimulated cAMP generation in cardiomyocytes from transgenic mice with cardiac-restricted overexpression of CTGF (Tg-CTGF) or cardiomyocytes pretreated with recombinant human CTGF (rec-hCTGF) revealed marked reduction of both ß1-AR and ß2-AR responsiveness. Consistently, ventricular muscle strips from Tg-CTGF mice stimulated with isoproterenol displayed attenuation of maximal inotropic responses. However, no differences of maximal inotropic responses of myocardial fibers from Tg-CTGF mice and nontransgenic littermate control (NLC) mice were discerned when stimulated with supramaximal concentrations of dibutyryl-cAMP, indicating preserved downstream responsiveness to cAMP. Congruent with a mechanism of desensitization of ß-ARs, mRNA and protein levels of G protein-coupled receptor kinase 5 (GRK5) were found isoform-selective upregulated in both cardiomyocytes from Tg-CTGF mice and cardiomyocytes exposed to rec-hCTGF. Corroborating a mechanism of GRK5 in CTGF-mediated control of ß-AR sensitivity, Chinese hamster ovary cells pretreated with rec-hCTGF displayed increased agonist- and biased ligand-stimulated ß-arrestin binding to ß-ARs. Despite increased sensitivity of cardiomyocytes from GRK5-knockout (KO) mice to ß-adrenergic agonists, pretreatment of GRK5-KO cardiomyocytes with rec-hCTGF, as opposed to cardiomyocytes from wild-type mice, did not alter ß-AR responsiveness. Finally, Tg-CTGF mice subjected to chronic exposure (14 days) to isoproterenol revealed blunted myocardial hypertrophy and preserved cardiac function versus NLC mice. In conclusion, this study uncovers a novel mechanism controlling ß-AR responsiveness in cardiomyocytes involving CTGF-mediated regulation of GRK5.

Evaluation of gene and cell-based therapies for cardiac regeneration.

Although the treatment of acute myocardial infarction has improved considerably and the mortality rate is reduced, patients who survive may develop loss of cardiomyocytes, scar formation, ventricular remodeling, and ultimately heart failure. The treatment of the most severe types of heart failure is heart transplantation, but this therapeutic intervention is not available for a large number of patients due to a shortage of donor hearts. Since current pharmacological and interventional approaches are unsuccessful to regenerate infarcted myocardium, new approaches like gene- or cell-based therapies are tested to prevent loss of cardiac tissue, enhance angiogenesis, and to reduce left ventricular remodeling. Exciting and promising data on laboratory animals have moved the field rapidly into clinical trials. Although several clinical trials proved the safety and feasibility of using gene- and cell-based therapies, many challenges remain before large-scale novel treatment modules will be available. The purpose of this review is to summarize the key findings of larger, randomized clinical trials in cardiovascular medicine using both gene and cell-based therapy, and to emphasize the most significant questions that emerged from the clinical experience so far, such as the optimal gene or cell type to be used, the ideal delivery route, and for DNA the ideal delivery system. Understanding the mechanisms of gene- and cell-based therapies is essential for designing the next phase clinical studies in the field of regenerative medicine.

The NLRP3 inflammasome is up-regulated in cardiac fibroblasts and mediates myocardial ischaemia-reperfusion injury.

AIMS: Nucleotide-binding oligomerization domain-Like Receptor with a Pyrin domain 3 (NLRP3) is considered necessary for initiating a profound sterile inflammatory response. NLRP3 forms multi-protein complexes with Apoptosis-associated Speck-like protein containing a Caspase recruitment domain (ASC) and Caspase-1, which activate pro-interleukin-1ß (IL-1ß) and pro-IL-18. The role of NLRP3 in cardiac cells is not known. Thus, we investigated the expression and function of NLRP3 during myocardial ischaemia. METHODS AND RESULTS: Myocardial infarction (MI) was induced in adult C57BL/6 mice and Wistar rats by ligation of the coronary artery. A marked increase in NLRP3, IL-1ß, and IL-18 mRNA expression was found in the left ventricle after MI, primarily located to myocardial fibroblasts. In vitro studies in cells from adult mice showed that myocardial fibroblasts released IL-1ß and IL-18 when primed with lipopolysaccharide and subsequently exposed to the danger signal adenosine triphosphate, a molecule released after tissue damage during MI. When hearts were isolated from NLRP3-deficient mice, perfused and subjected to global ischaemia and reperfusion, a marked improvement of cardiac function and reduction of hypoxic damage was found compared with wild-type hearts. This was not observed in ASC-deficient hearts, potentially reflecting a protective role of other ASC-dependent inflammasomes or inflammasome-independent effects of NLRP3. CONCLUSION: This study shows that the NLRP3 inflammasome is up-regulated in myocardial fibroblasts post-MI, and may be a significant contributor to infarct size development during ischaemia-reperfusion.