Rolle des Cystein-reichen LIM-Proteins 4 (CRP4) für die cGMP-modulierte Pathobiologie myokardialer Remodelingprozesse

Kardiale Hypertrophie ist zumeist eine Folgeerkrankung aufgrund einer Überbelastung des Arbeitsmyokards durch Bluthochdruck, Herzklappendefekten sowie neurohumoraler Stimulation. In diesem Kontext signalisiert das Neurohormon Angiotensin II (Ang II) als Komponente des lokalen Renin-Angiotensin-Aldosteron-Systems (RAAS) in Herzmuskelzellen über Gαq-gekoppelte AT1-Rezeptoren (AT1R) und ist hierbei ein entscheidender Faktor der pro-hypertrophen und pro-fibrotischen myokardialen Gewebeumbauprozesse. Darüber hinaus führen Ang II/AT1R über vasokonstriktorische und renale Wirkungen zu einer Blutdruckerhöhung und damit zu krankhaftem Herzmuskelwachstum. Ein wichtiges endogenes System zur Gegenregulation und Funktionserhaltung des Herzens ist der cGMP/cGKI-Signalweg. In der vorliegenden Arbeit wurde eine mögliche Protektion des cGMP/cGKI-Systems sowohl bei genetisch als auch pharmakologisch intensivierter Ang II-Signaltransduktion im murinen Myokard untersucht. Hierbei konnte gezeigt werden, dass Komponenten dieses Signalwegs aufgrund der amplifizierten myozytären Ang II-Signaltransduktion hochreguliert waren, was wiederum für die schützende Bedeutung von kardialem cGMP/cGKI spricht. Bislang ist jedoch vergleichsweise wenig über die der cGKI (cGMP-abhängige Proteinkinase I) nachgeschalteten kardialen Substrate bekannt. Im Rahmen dieser Arbeit konnte das Cystein-reiche LIM-Protein 4 (CRP4) als neues cGKI-Substrat im Herzmuskel identifiziert werden. CRP4 gehört zur Klasse der LIM-Proteine, die meist als Adapterproteine fungieren und somit die Integrität, Stabilität und Funktion von Proteinkomplexen modulieren. Um die möglichen Funktionen von kardialem CRP4 zu analysieren, wurden in der vorliegenden Arbeit CRP4-Wildtyp und CRP4-Knockout Wurfgeschwister verglichen. Im Vordergrund standen zunächst Untersuchungen zur Expression von CRP4 im Myokard, zu Herzfunktion und Morphologie sowie die Identifikation von CRP4-Interaktionspartnern unter physiologischen Bedingungen und bei neurohumoral induzierter Myokardhypertrophie. Auf diese Weise konnte CRP4 erstmalig als neue Komponente eines grundlegenden endogenen Schutzmechanismus bei Ang II-induzierter myokardialen Remodelingprozessen beschrieben werden. Die Deletion von CRP4 resultiert in diesem Zusammenhang außerdem in zahlreichen Veränderungen in der Expression und Funktion zentraler Komponenten der cGMP/cGKI-Signalkaskade. Der Nachweis von cGKI/CRP4-Proteinkomplexen im hypertrophen Myokard bestätigt letztlich, dass CRP4 an den protektiven Wirkungen des kardialen cGMP/cGKI-Signalwegs beteiligt ist

Rolle des Cystein-reichen LIM-Proteins 4 (CRP4) für die cGMP-modulierte Pathobiologie myokardialer Remodelingprozesse

Kardiale Hypertrophie ist zumeist eine Folgeerkrankung aufgrund einer Überbelastung des Arbeitsmyokards durch Bluthochdruck, Herzklappendefekten sowie neurohumoraler Stimulation. In diesem Kontext signalisiert das Neurohormon Angiotensin II (Ang II) als Komponente des lokalen Renin-Angiotensin-Aldosteron-Systems (RAAS) in Herzmuskelzellen über Gαq-gekoppelte AT1-Rezeptoren (AT1R) und ist hierbei ein entscheidender Faktor der pro-hypertrophen und pro-fibrotischen myokardialen Gewebeumbauprozesse. Darüber hinaus führen Ang II/AT1R über vasokonstriktorische und renale Wirkungen zu einer Blutdruckerhöhung und damit zu krankhaftem Herzmuskelwachstum. Ein wichtiges endogenes System zur Gegenregulation und Funktionserhaltung des Herzens ist der cGMP/cGKI-Signalweg. In der vorliegenden Arbeit wurde eine mögliche Protektion des cGMP/cGKI-Systems sowohl bei genetisch als auch pharmakologisch intensivierter Ang II-Signaltransduktion im murinen Myokard untersucht. Hierbei konnte gezeigt werden, dass Komponenten dieses Signalwegs aufgrund der amplifizierten myozytären Ang II-Signaltransduktion hochreguliert waren, was wiederum für die schützende Bedeutung von kardialem cGMP/cGKI spricht. Bislang ist jedoch vergleichsweise wenig über die der cGKI (cGMP-abhängige Proteinkinase I) nachgeschalteten kardialen Substrate bekannt. Im Rahmen dieser Arbeit konnte das Cystein-reiche LIM-Protein 4 (CRP4) als neues cGKI-Substrat im Herzmuskel identifiziert werden. CRP4 gehört zur Klasse der LIM-Proteine, die meist als Adapterproteine fungieren und somit die Integrität, Stabilität und Funktion von Proteinkomplexen modulieren. Um die möglichen Funktionen von kardialem CRP4 zu analysieren, wurden in der vorliegenden Arbeit CRP4-Wildtyp und CRP4-Knockout Wurfgeschwister verglichen. Im Vordergrund standen zunächst Untersuchungen zur Expression von CRP4 im Myokard, zu Herzfunktion und Morphologie sowie die Identifikation von CRP4-Interaktionspartnern unter physiologischen Bedingungen und bei neurohumoral induzierter Myokardhypertrophie. Auf diese Weise konnte CRP4 erstmalig als neue Komponente eines grundlegenden endogenen Schutzmechanismus bei Ang II-induzierter myokardialen Remodelingprozessen beschrieben werden. Die Deletion von CRP4 resultiert in diesem Zusammenhang außerdem in zahlreichen Veränderungen in der Expression und Funktion zentraler Komponenten der cGMP/cGKI-Signalkaskade. Der Nachweis von cGKI/CRP4-Proteinkomplexen im hypertrophen Myokard bestätigt letztlich, dass CRP4 an den protektiven Wirkungen des kardialen cGMP/cGKI-Signalwegs beteiligt ist

Detection of Leptospira DNA in urine and presence of specific antibodies in outdoor cats in Germany

Objectives Clinical manifestation of infection with Leptospira species in cats is rare. Nevertheless, cats can develop specific antibodies against the spirochetes after infection. In Canada, Taiwan and the USA it was recently demonstrated that naturally infected cats can also shed DNA from pathogenic Leptospira species in their urine, but the zoonotic potential of infected cats is still unclear. The objective of this study was to demonstrate if outdoor cats in Germany shed DNA from pathogenic Leptospira species in their urine. As a second aim, antibody prevalence was determined. Methods Two hundred and fifteen outdoor cats were prospectively recruited. Urine samples were tested by realtime PCR targeting the lipL32 gene of pathogenic Leptospira species. Antibody titres against eight serovars (Australis, Autumnalis, Bratislava, Canicola, Copenhageni, Grippotyphosa, Pomona, Saxkoebing) belonging to seven serogroups (Australis, Autumnalis, Canicola, Grippotyphosa, Icterohaemorrhagiae, Pomona, Sejroe) were determined by microscopic agglutination test. Results Urine samples from 7/215 cats (3.3%;95% confidence interval [CI] 0.9-5.7) were PCR-positive. Specific antibodies were detected in 35/195 cats (17.9%;95% CI: 12.5-23.3) with titres ranging from 1:100 to 1:6400. Australis, Bratislava and Grippotyphosa were the most common serovars. Conclusions and relevance Outdoor cats in Germany can shed DNA from pathogenic Leptospira species. Therefore, outdoor cats should be considered as a possible source of infection for dogs or humans. Further studies are needed to determine the role of Leptospira species as a cause of disease in cats

cGMP-Elevating Compounds and Ischemic Conditioning Provide Cardioprotection Against Ischemia and Reperfusion Injury via Cardiomyocyte-Specific BK Channels.

BACKGROUND: The nitric oxide-sensitive guanylyl cyclase/cGMP-dependent protein kinase type I signaling pathway can afford protection against the ischemia/reperfusion injury that occurs during myocardial infarction. Reportedly, voltage and Ca2+-activated K+ channels of the BK type are stimulated by cGMP/cGMP-dependent protein kinase type I, and recent ex vivo studies implicated that increased BK activity favors the survival of the myocardium at ischemia/reperfusion. It remains unclear, however, whether the molecular events downstream of cGMP involve BK channels present in cardiomyocytes or in other cardiac cell types. METHODS: Gene-targeted mice with a cardiomyocyte- or smooth muscle cell-specific deletion of the BK (CMBK or SMBK knockouts) were subjected to the open-chest model of myocardial infarction. Infarct sizes of the conditional mutants were compared with litter-matched controls, global BK knockout, and wild-type mice. Cardiac damage was assessed after mechanical conditioning or pharmacological stimulation of the cGMP pathway and by using direct modulators of BK. Long-term outcome was studied with respect to heart functions and cardiac fibrosis in a chronic myocardial infarction model. RESULTS: Global BK knockouts and CMBK knockouts, in contrast with SMBK knockouts, exhibited significantly larger infarct sizes compared with their respective controls. Ablation of CMBK resulted in higher serum levels of cardiac troponin I and elevated amounts of reactive oxygen species, lower phosphorylated extracellular receptor kinase and phosphorylated AKT levels and an increase in myocardial apoptosis. Moreover, CMBK was required to allow beneficial effects of both nitric oxide-sensitive guanylyl cyclase activation and inhibition of the cGMP-degrading phosphodiesterase-5, ischemic preconditioning, and postconditioning regimens. To this end, after 4 weeks of reperfusion, fibrotic tissue increased and myocardial strain echocardiography was significantly compromised in CMBK-deficient mice. CONCLUSIONS: Lack of CMBK channels renders the heart more susceptible to ischemia/reperfusion injury, whereas the pathological events elicited by ischemia/reperfusion do not involve BK in vascular smooth muscle cells. BK seems to permit the protective effects triggered by cinaciguat, riociguat, and different phosphodiesterase-5 inhibitors and beneficial actions of ischemic preconditioning and ischemic postconditioning by a mechanism stemming primarily from cardiomyocytes. This study establishes mitochondrial CMBK channels as a promising target for limiting acute cardiac damage and adverse long-term events that occur after myocardial infarction

KCNMA1 Encoded Cardiac BK Channels Afford Protection against Ischemia-Reperfusion Injury

Mitochondrial potassium channels have been implicated in myocardial protection mediated through pre-/postconditioning. Compounds that open the Ca2+-and voltage-activated potassium channel of big-conductance (BK) have a pre-conditioninglike effect on survival of cardiomyocytes after ischemia/reperfusion injury. Recently, mitochondrial BK channels (mitoBKs) in cardiomyocytes were implicated as infarct-limiting factors that derive directly from the KCNMA1 gene encoding for canonical BKs usually present at the plasma membrane of cells. However, some studies challenged these cardio-protective roles of mitoBKs. Herein, we present electrophysiological evidence for paxilline- and NS11021-sensitive BK-mediated currents of 190 pS conductance in mitoplasts from wild-type but not BK-/- cardiomyocytes. Transmission electron microscopy of BK-/- ventricular muscles fibres showed normal ultra-structures and matrix dimension, but oxidative phosphorylation capacities at normoxia and upon re-oxygenation after anoxia were significantly attenuated in BK-/- permeabilized cardiomyocytes. In the absence of BK, post-anoxic reactive oxygen species (ROS) production from cardiomyocyte mitochondria was elevated indicating that mitoBK fine-tune the oxidative state at hypoxia and reoxygenation. Because ROS and the capacity of the myocardium for oxidative metabolism are important determinants of cellular survival, we tested BK-/- hearts for their response in an ex-vivo model of ischemia/reperfusion (I/R) injury. Infarct areas, coronary flow and heart rates were not different between wild-type and BK-/- hearts upon I/R injury in the absence of ischemic pre-conditioning (IP),but differed upon IP. While the area of infarction comprised 28 +/- 3% of the area at risk in wild-type, it was increased to 58 +/- 5% in BK-/- hearts suggesting that BK mediates the beneficial effects of IP. These findings suggest that cardiac BK channels are important for proper oxidative energy supply of cardiomyocytes at normoxia and upon re-oxygenation after prolonged anoxia and that IP might indeed favor survival of the myocardium upon I/R injury in a BK-dependent mode stemming from both mitochondrial post-anoxic ROS modulation and non-mitochondrial localizations

Title Page Sildenafil does not prevent heart hypertrophy and fibrosis induced by cardiomyocyte AT 1 R signaling

List of non-standard abbreviations: cGMP-dependent protein kinase type I (cGKI); protein kinase G (PKG); natriuretic peptides (NPs), cyclic guanosine-3´,5´-monophosphate (cGMP); nitric oxide (NO); cyclic adenosine-3´,5´-monophosphate (cAMP); Angiotensin II (AngII); Angiotensin II receptor type 1 (AT 1 R); sildenafil (SIL); cardiomyocyte (CM), smooth muscle cell (SMC); angiotensin converting enzyme (ACE), cGKIβ rescue mice (βRM), phosphodiesterase (PDE); Förster resonance energy transfer (FRET); 3-isobutyl-1-methylxanthine (IBMX), C-type natriuretic peptide (CNP). Section assignment: Cardiovascular Number of words: 4141 (excluding abstract, material and methods, reference list and figure legends) Number of words (abstract): 250 Number of references: 67 Page count: 37 JPET#226092 3 Abstract Analyses of several mouse models imply that the phosphodiesterase 5 (PDE5) inhibitor sildenafil (SIL), via increasing cyclic guanosine-3',5'-monophosphate (cGMP), affords protection against angiotensin II (AngII) stimulated cardiac remodeling. However, it is unclear which cell types are involved in these beneficial effects, because AngII may exert its adverse effects by modulating multiple reno-vascular and cardiac functions via AngII type 1 receptors (AT 1 R). To test the hypothesis that SIL/cGMP oppose cardiac stress provoked by amplified AngII/AT 1 R directly in cardiomyocytes (CMs), we studied transgenic mice with CM-specific overexpression of the AT 1 R under the control of the α -myosin-heavy chain promoter (αMHC-AT 1 R tg/+ ). The extent of cardiac growth was assessed in absence or presence of SIL and defined by referring changes in heart-weight to body-weight or tibia length. Hypertrophic marker genes, extracellular matrix-regulating factors and expression patterns of fibrosis markers were examined in α MHC-AT 1 R tg/+ ventricles (±SIL) and corroborated by investigating different components of the natriuretic peptide (NP)/PDE5/cGMP pathway as well as cardiac functions. cGMP levels in heart lysates and intact CMs were measured by competitive immunoassays and FRET. We find higher cardiac and CM cGMP levels and up-regulation of the cGMP-dependent protein kinase I (cGKI) with AT 1 R over-expression. However, even a prolonged SIL treatment regimen did not limit the progressive CM growth, fibrosis or decline in cardiac functions in the αMHC-AT 1 R tg/+ model suggesting that SIL does not interfere with the pathogenic actions of amplified AT 1 R signaling in CMs. Hence, the cardiac/non-cardiac cells involved in the cross-talk between SIL-sensitive PDE activity and AngII/AT 1 R need to be identified

Analysis of mass spectrometry data from the secretome of an explant model of articular cartilage exposed to pro-inflammatory and anti-inflammatory stimuli using machine learning

Background: Osteoarthritis (OA) is an inflammatory disease of synovial joints involving the loss and degeneration of articular cartilage. The gold standard for evaluating cartilage loss in OA is the measurement of joint space width on standard radiographs. However, in most cases the diagnosis is made well after the onset of the disease, when the symptoms are well established. Identification of early biomarkers of OA can facilitate earlier diagnosis, improve disease monitoring and predict responses to therapeutic interventions. Methods: This study describes the bioinformatic analysis of data generated from high throughput proteomics for identification of potential biomarkers of OA. The mass spectrometry data was generated using a canine explant model of articular cartilage treated with the pro-inflammatory cytokine interleukin 1 β (IL-1β). The bioinformatics analysis involved the application of machine learning and network analysis to the proteomic mass spectrometry data. A rule based machine learning technique, BioHEL, was used to create a model that classified the samples into their relevant treatment groups by identifying those proteins that separated samples into their respective groups. The proteins identified were considered to be potential biomarkers. Protein networks were also generated; from these networks, proteins pivotal to the classification were identified. Results: BioHEL correctly classified eighteen out of twenty-three samples, giving a classification accuracy of 78.3% for the dataset. The dataset included the four classes of control, IL-1β, carprofen, and IL-1β and carprofen together. This exceeded the other machine learners that were used for a comparison, on the same dataset, with the exception of another rule-based method, JRip, which performed equally well. The proteins that were most frequently used in rules generated by BioHEL were found to include a number of relevant proteins including matrix metalloproteinase 3, interleukin 8 and matrix gla protein. Conclusions: Using this protocol, combining an in vitro model of OA with bioinformatics analysis, a number of relevant extracellular matrix proteins were identified, thereby supporting the application of these bioinformatics tools for analysis of proteomic data from in vitro models of cartilage degradation

Antimicrobial Resistance, Serologic and Molecular Characterization of <i>E. coli</i> Isolated from Calves with Severe or Fatal Enteritis in Bavaria, Germany

Worldwide, enterotoxigenic Escherichia coli (ETEC) cause neonatal diarrhea and high mortality rates in newborn calves, leading to great economic losses. In Bavaria, Germany, no recent facts are available regarding the prevalence of virulence factors or antimicrobial resistance of ETEC in calves. Antimicrobial susceptibility of 8713 E. coli isolates obtained from 7358 samples of diseased or deceased diarrheic calves were investigated between 2015 to 2019. Considerably high rates of 84.2% multidrug-resistant and 15.8% extensively drug-resistant isolates were detected. The resistance situation of the first, second and third line antimicrobials for the treatment, here amoxicillin-clavulanate, enrofloxacin and trimethoprim-sulfamethoxazole, is currently acceptable with mean non-susceptibility rates of 28.1%, 37.9% and 50.0% over the investigated 5-year period. Furthermore, the ETEC serotypes O101:K28, O9:K35, O101:K30, O101:K32, O78:K80, O139:K82, O8:K87, O141:K85 and O147:K89, as well as the virulence factors F17, F41, F5, ST-I and stx1 were identified in a subset of samples collected in 2019 and 2020. The substantially high rates of multi- and extensively drug-resistant isolates underline the necessity of continuous monitoring regarding antimicrobial resistance to provide reliable prognoses and adjust recommendations for the treatment of bacterial infections in animals

Phenotypic characterization and whole genome analysis of extended-spectrum beta-lactamase-producing bacteria isolated from dogs in Germany.

Asymptomatic colonization with extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae has been described for humans, various mammal species, and birds. Here, antimicrobial resistant bacteria were recovered from dog feces originating in Germany, Kosovo, Afghanistan, Croatia, and Ukraine, with a subset of mostly E. coli isolates obtained from a longitudinal collection over twelve months. In vitro antimicrobial resistance testing revealed various patterns of resistance against single or all investigated beta-lactam antibiotics, with none of the 101 isolates resistant against two tested carbapenem antibiotics. Whole genome sequence analysis revealed bacteria species-specific patterns for 23 antimicrobial resistance coding DNA sequences (CDS) that were unapparent from the in vitro analysis alone. Phylogenetic analysis of single nucleotide polymorphisms (SNP) revealed clonal bacterial isolates originating from different dogs, suggesting transmission between dogs in the same community. However, individual resistant E. coli clones were not detected over a period longer than seven days. Multi locus sequence typing (MLST) of 85 E. coli isolates revealed 31 different sequence types (ST) with an accumulation of ST744 (n = 9), ST10 (n = 8), and ST648 (n = 6), although the world-wide hospital-associated CTX-M beta-lactamase producing ST131 was not detected. Neither the antimicrobial resistance CDSs patterns nor the phylogenetic analysis revealed an epidemiological correlation among the longitudinal isolates collected from a period longer than seven days. No genetic linkage could be associated with the geographic origin of isolates. In conclusion, healthy dogs frequently carry ESBL-producing bacteria, independent to prior treatment, which may be transmitted between individual dogs of the same community. Otherwise, these antimicrobial resistant bacteria share few commonalities, making their presence eerily unpredictable