Natalie Burkard

Conditional Neuronal Nitric Oxide Synthase Overexpression Impairs Myocardial Contractility

The role of the neuronal NO synthase (nNOS or NOS1) enzyme in the control of cardiac function still remains unclear. Results from nNOS−/− mice or from pharmacological inhibition of nNOS are contradictory and do not pay tribute to the fact that probably spatial confinement of the nNOS enzyme is of major importance. We hypothesize that the close proximity of nNOS and certain effector molecules like L-type Ca2+-channels has an impact on myocardial contractility. To test this, we generated a new transgenic mouse model allowing conditional, myocardial specific nNOS overexpression. Western blot analysis of transgenic nNOS overexpression showed a 6-fold increase in nNOS protein expression compared with noninduced littermates (n=12; P<0.01). Measuring of total NOS activity by conversion of [3H]-l-arginine to [3H]-l-citrulline showed a 30% increase in nNOS overexpressing mice (n=18; P<0.05). After a 2 week induction, nNOS overexpression mice showed reduced myocardial contractility. In vivo examinations of the nNOS overexpressing mice revealed a 17±3% decrease of +dp/dtmax compared with noninduced mice (P<0.05). Likewise, ejection fraction was reduced significantly (42% versus 65%; n=15; P<0.05). Interestingly, coimmunoprecipitation experiments indicated interaction of nNOS with SR Ca2+ATPase and additionally with L-type Ca2+- channels in nNOS overexpressing animals. Accordingly, in adult isolated cardiac myocytes, ICa,L density was significantly decreased in the nNOS overexpressing cells. Intracellular Ca2+-transients and fractional shortening in cardiomyocytes were also clearly impaired in nNOS overexpressing mice versus noninduced littermates. In conclusion, conditional myocardial specific overexpression of nNOS in a transgenic animal model reduced myocardial contractility. We suggest that nNOS might suppress the function of L-type Ca2+-channels and in turn reduces Ca2+-transients which accounts for the negative inotropic effect.

Targeted Proteolysis Sustains Calcineurin Activation

Background—Calcineurin (CnA) is important in the regulation of myocardial hypertrophy. We demonstrated that targeted proteolysis of the CnA autoinhibitory domain under pathological myocardial workload leads to increased CnA activity in human myocardium. Here, we investigated the proteolytic mechanism leading to activation of CnA. Methods and Results—In patients with diseased myocardium, we found strong nuclear translocation of CnA. In contrast, in normal human myocardium, there was a cytosolic distribution of CnA. Stimulation of rat cardiomyocytes with angiotensin (Ang) II increased calpain activity significantly (433±11%; P<0.01; n=6) and caused proteolysis of the autoinhibitory domain of CnA. Inhibition of calpain by a membrane-permeable calpain inhibitor prevented proteolysis. We identified the cleavage site of calpain in the human CnA sequence at amino acid 424. CnA activity was increased after Ang II stimulation (310±29%; P<0.01; n=6) and remained high after removal of Ang II (214±17%; P<0.01; n=6). Addition of a calpain inhibitor to the medium decreased CnA activity (110±19%; P=NS; n=6) after removal of Ang II. Ang II stimulation of cardiomyocytes also translocated CnA into the nucleus as demonstrated by immunohistochemical staining and transfection assays with GFP-tagged CnA. Calpain inhibition and therefore suppression of calpain-mediated proteolysis of CnA enabled CnA exit from the nucleus. Conclusions—Ang II stimulation of cardiomyocytes increased calpain activity, leading to proteolysis of the autoinhibitory domain of CnA. This causes an increase in CnA activity and results in nuclear translocation of CnA. Loss of the autoinhibitory domain renders CnA constitutively nuclear and active, even after removal of the hypertrophic stimulus.

AT2 receptor activation regulates myocardial eNOS expression via the calcineurin–NF-AT pathway

The role of AT2‐receptors has recently been subject of considerable debate. We investigated the influence of AT2‐stimulation/inhibition on myocardial endothelial NO‐synthase (eNOS, NOS‐III) promoter activity and eNOS protein expression.

Protective effects of sphingosine-1-phosphate receptor agonist treatment after myocardial ischaemia-reperfusion.

AIMS Several experimental studies have demonstrated protection against cardiac ischaemia-reperfusion injury achieved by pre-treatment with exogenous sphingosine-1-phosphate (S1P). We tested the hypothesis that pharmacological S1P receptor agonists improve recovery of function when applied with reperfusion. METHODS AND RESULTS Isolated rat cardiomyocytes were stimulated with exogenous S1P, the selective S1P1 receptor agonist SEW2871, or the S1P1/3 receptor agonist FTY720. Western blot analysis was performed to analyse downstream signalling pathways. Ischaemia-reperfusion studies were conducted in rat cardiomyocytes, isolated Langendorff-perfused rat hearts, and in human myocardial muscle strip preparations to evaluate the effect of S1P receptor agonists on cell death and recovery of mechanical function. All S1P receptor agonists were able to activate Akt. This was associated with transactivation of the epidermal growth factor receptor. In isolated cardiomyocytes, selective stimulation of the S1P1 receptor by SEW2871 induced protection against cell death when administered either before or after ischaemia-reperfusion. In isolated rat hearts, treatment with FTY720 during reperfusion attenuated the rise in left ventricular end-diastolic pressure (LVEDP) and improved the recovery of left ventricular developed pressure without limiting infarct size. However, selective S1P1 receptor stimulation did not improve functional recovery but rather increased LVEDP. Additional experiments employing a human myocardial ischaemia-reperfusion model also demonstrated improved functional recovery induced by FTY720 treatment during reperfusion. CONCLUSION Pharmacological S1P receptor agonists have distinct effects on ischaemia-reperfusion injury. Their efficacy when applied during reperfusion makes them potential candidates for pharmaceutical postconditioning therapy after cardiac ischaemia.

Conditional Overexpression of Neuronal Nitric Oxide Synthase Is Cardioprotective in Ischemia/Reperfusion

Background— We previously demonstrated that conditional overexpression of neuronal nitric oxide synthase (nNOS) inhibited L-type Ca2+ channels and decreased myocardial contractility. However, nNOS has multiple targets within the cardiac myocyte. We now hypothesize that nNOS overexpression is cardioprotective after ischemia/reperfusion because of inhibition of mitochondrial function and a reduction in reactive oxygen species generation. Methods and Results— Ischemia/reperfusion injury in wild-type mice resulted in nNOS accumulation in the mitochondria. Similarly, transgenic nNOS overexpression caused nNOS abundance in mitochondria. nNOS translocation into the mitochondria was dependent on heat shock protein 90. Ischemia/reperfusion experiments in isolated hearts showed a cardioprotective effect of nNOS overexpression. Infarct size in vivo was also significantly reduced. nNOS overexpression also caused a significant increase in mitochondrial nitrite levels accompanied by a decrease of cytochrome c oxidase activity. Accordingly, O2 consumption in isolated heart muscle strips was decreased in nNOS-overexpressing nNOS+/&agr;MHC-tTA+ mice already under resting conditions. Additionally, we found that the reactive oxygen species concentration was significantly decreased in hearts of nNOS-overexpressing nNOS+/&agr;MHC-tTA+ mice compared with noninduced nNOS+/&agr;MHC-tTA+ animals. Conclusion— We demonstrated that conditional transgenic overexpression of nNOS resulted in myocardial protection after ischemia/reperfusion injury. Besides a reduction in reactive oxygen species generation, this might be caused by nitrite-mediated inhibition of mitochondrial function, which reduced myocardial oxygen consumption already under baseline conditions.

Impact of imaging landmark on the risk of MRI‐related heating near implanted medical devices like cardiac pacemaker leads

Implanted medical devices such as cardiac pacemakers pose a potential hazard in magnetic resonance imaging. Electromagnetic fields have been shown to cause severe radio frequency‐induced tissue heating in some cases. Imaging exclusion zones have been proposed as an instrument to reduce patient risk. The purpose of this study was to further assess the impact of the imaging landmark on the risk for unintended implant heating by measuring the radio frequency‐induced electric fields in a body phantom under several imaging conditions at 1.5T. The results show that global radio frequency‐induced coupling is highest with the torso centered along the superior–inferior direction of the transmit coil. The induced E‐fields inside the body shift when changing body positioning, reducing both global and local radio frequency coupling if body and/or conductive implant are moved out from the transmit coil center along the z‐direction. Adequate selection of magnetic resonance imaging landmark can significantly reduce potential hazards in patients with implanted medical devices. Magn Reson Med, 2010.

Soluble VE-cadherin is involved in endothelial barrier breakdown in systemic inflammation and sepsis

AIMS Microvascular endothelial barrier breakdown in sepsis precedes organ failure and death in patients. We tested the hypothesis that the formation of endothelium-derived soluble vascular endothelial (VE)-cadherin fragments (sVE-cadherin) is involved in inflammation-induced endothelial barrier disruption. METHODS AND RESULTS Incubation of human dermal microvascular endothelial cells (HDMEC) with tumour necrosis factor-α (TNF-α) and bacterial lipopolysaccharide (LPS) led to endothelial barrier disruption which correlated with significantly increased sVE-cadherin at a size of ∼90 kDa in cell culture supernatants. Inhibition of the VE-cadherin-cleaving disintegrin and metalloproteinase ADAM10 using GI254023X attenuated inflammation-induced formation of sVE-cadherin and endothelial barrier disruption, suggesting ADAM10-mediated shedding as a mechanism underlying sVE-cadherin release. Formation of VE-cadherin fragments at 90 and 110 kDa was observed when recombinant VE-cadherin (rVE-cadherin) was digested with recombinant ADAM10. Mass spectrometry of the VE-cadherin fragments showed that they originated from cleavage of the extracelluar domain and thereby several cleavage sites of ADAM10 were identified. Atomic force microscopy measurements demonstrated that cell culture supernatants containing sVE-cadherin and application of rVE-cadherin blocked VE-cadherin binding. Accordingly rVE-cadherin dose-dependently led to loss of endothelial barrier functions in HDMEC monolayers. Finally, in patients suffering from severe sepsis or septic shock with clinical signs of a microvascular leackage, serum levels of sVE-cadherin were significantly increased. CONCLUSION Taken together, formation of sVE-cadherin is associated and contributes to inflammation-induced breakdown of endothelial barrier functions by inhibition of VE-cadherin binding. The underlying mechanism of VE-cadherin cleavage involves ADAM10 and appears to be of clinical relevance since sVE-cadherin was augmented in patients with severe sepsis.

Feasibility of Contrast-Enhanced and Nonenhanced MRI for Intraprocedural and Postprocedural Lesion Visualization in Interventional Electrophysiology Animal Studies and Early Delineation of Isthmus Ablation Lesions in Patients With Typical Atrial Flutter

Background— Imaging of myocardial ablation lesions during electrophysiology procedures would enable superior guidance of interventions and immediate identification of potential complications. The aim of this study was to establish clinically suitable MRI-based imaging techniques for intraprocedural lesion visualization in interventional electrophysiology. Methods and Results— Interventional electrophysiology was performed under magnetic resonance guidance in an animal model, using a custom setup including magnetic resonance–conditional catheters. Various pulse sequences were explored for intraprocedural lesion visualization after radiofrequency ablation. The developed visualization techniques were then used to investigate lesion formation in patients immediately after ablation of atrial flutter. The animal studies in 9 minipigs showed that gadolinium-DTPA–enhanced T1-weighted and nonenhanced T2-weighted pulse sequences are particularly suitable for lesion visualization immediately after radiofrequency ablation. MRI-derived lesion size correlated well with autopsy (R2=0.799/0.709 for contrast-enhanced/nonenhanced imaging). Non–contrast agent–enhanced techniques were suitable for repetitive lesion visualization during electrophysiological interventions, thus allowing for intraprocedural monitoring of ablation success. The patient studies in 24 patients with typical atrial flutter several minutes to hours after cavotricuspid isthmus ablation confirmed the results from the animal experiments. Therapeutic lesions could be visualized in all patients using contrast-enhanced and also nonenhanced MRI with high contrast-to-noise ratio (94.6±35.2/111.1±32.6 versus 48.0±29.0/68.0±37.3 for ventricular/atrial lesions and contrast-enhanced versus nonenhanced imaging). Conclusions— MRI allows for precise lesion visualization in electrophysiological interventions just minutes after radiofrequency ablation. Nonenhanced T2-weighted MRI is particularly feasible for intraprocedural delineation of lesion formation as lesions are detectable within minutes after radiofrequency delivery and imaging can be repeated during interventions.

Glial cell line-derived neurotrophic factor promotes barrier maturation and wound healing in intestinal epithelial cells in vitro.

Recent data suggest that neurotrophic factors from the enteric nervous system are involved in intestinal epithelial barrier regulation. In this context the glial cell line-derived neurotrophic factor (GDNF) was shown to affect gut barrier properties in vivo directly or indirectly by largely undefined processes in a model of inflammatory bowel disease (IBD). We further investigated the potential role and mechanisms of GDNF in the regulation of intestinal barrier functions. Immunostaining of human gut specimen showed positive GDNF staining in enteric neuronal plexus and in enterocytes. In Western blots of the intestinal epithelial cell lines Caco2 and HT29B6, significant amounts of GDNF were detected, suggesting that enterocytes represent an additional source of GDNF. Application of recombinant GDNF on Caco2 and HT29B6 cells for 24 h resulted in significant epithelial barrier stabilization in monolayers with immature barrier functions. Wound-healing assays showed a significantly faster closure of the wounded areas after GDNF application. GDNF augmented cAMP levels and led to significant inactivation of p38 MAPK in immature cells. Activation of p38 MAPK signaling by SB-202190 mimicked GDNF-induced barrier maturation, whereas the p38 MAPK activator anisomycin blocked GDNF-induced effects. Increasing cAMP levels had adverse effects on barrier maturation, as revealed by permeability measurements. However, increased cAMP augmented the proliferation rate in Caco2 cells, and GDNF-induced proliferation of epithelial cells was abrogated by the PKA inhibitor H89. Our data show that enterocytes represent an additional source of GDNF synthesis. GDNF contributes to wound healing in a cAMP/PKA-dependent manner and promotes barrier maturation in immature enterocytes cells by inactivation of p38 MAPK signaling.

The glial cell-line derived neurotrophic factor: a novel regulator of intestinal barrier function in health and disease

Regulation of the intestinal epithelial barrier is a differentiated process, which is profoundly deranged in inflammatory bowel diseases. Recent data provide evidence that the glial cell line-derived neurotrophic factor (GDNF) is critically involved in intestinal epithelial wound healing and barrier maturation and exerts antiapoptotic effects under certain conditions. Furthermore, not only the enteric nervous system, but also enterocytes synthesize GDNF in significant amounts, which points to a potential para- or autocrine signaling loop between enterocytes. Apart from direct effects of GDNF on enterocytes, an immunomodulatory role of this protein has been previously assumed because of a significant reduction of inflammation in a model of chronic inflammatory bowel disease after application of GDNF. In this review we summarize the current knowledge of GDNF on intestinal epithelial barrier regulation and discuss the novel role for GDNF as a regulator of intestinal barrier functions in health and disease.