On the design and simulation of an airlift loop bioreactor with microbubble generation by fluidic oscillation

Microbubble generation by a novel fluidic oscillator driven approach is analyzed, with a view to identifying the key design elements and their differences from standard approaches to airlift loop bioreactor design. The microbubble generation mechanism has been shown to achieve high mass transfer rates by the decrease of the bubble diameter, by hydrodynamic stabilization that avoids coalescence increasing the bubble diameter, and by longer residence times offsetting slower convection. The fluidic oscillator approach also decreases the friction losses in pipe networks and in nozzles/diffusers due to boundary layer disruption, so there is actually an energetic consumption savings in using this approach over steady flow. These dual advantages make the microbubble generation approach a promising component of a novel airlift loop bioreactor whose design is presented here. The equipment, control system for flow and temperature, and the optimization of the nozzle bank for the gas distribution system are presented. (C) 2009 The Institution of Chemical Engineers. Published by Elsevier B.V All rights reserved

GPCR-dependent pathways promote nanodomain-specific cAMP signaling in human cardiomyocytes, which is severely remodeled in atrial fibrillation

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft, German Centre for Cardiovascular Research (DZHK) Background Cardiac arrhythmias, such as atrial fibrillation (AF), have been linked to the remodeling of membrane receptors and alterations in cAMP-dependent regulation of calcium handling mechanisms. For instance, decreased L-type calcium channel (LTCC) current density but upregulated ryanodine receptor 2 (RyR2) are major hallmarks of AF. Furthermore, adenosine A2A receptor (A2AR) stimulation increases calcium waves without affecting LTCC and serotonin (5-HT) receptors activation exerts stronger control over myofilaments than over RyR2 function. However, up to date no study has elucidated how the increase on cAMP upon different G-protein-coupled receptors (GPCR) stimulation can lead to different physiological compartmentalized responses. The aim of this study was to investigate the effects of various GPCRs on cAMP levels in different cellular compartments in human atrial myocytes from control patients in sinus rhythm (Ctl), and how these compartmentalized effects are altered in AF. Furthermore, alterations in downstream cAMP level control by phosphodiesterases (PDEs) between patient groups were investigated to further elucidate functional differences. Methods Atrial myocytes were isolated from tissues of 66 AF and 80 Ctl patients. Cells were then transduced with adenoviruses (Epac1-camps, pm-Epac1-camps and Epac1-JNC) and cultured for 48 hours to express the Förster-resonance energy transfer (FRET)-based cAMP sensor in the cytosolic, membrane and RyR2 nanodomains, respectively. FRET was then used to measure cAMP in 532 isolated human atrial myocytes. Stimulation with β-adrenergic agonist Isoprenaline (ISO, 100nM) was used and compared with 5-HT (100µM) and A2AR (with CGS, 200nM) stimulation. Additionally, a nonspecific PDE inhibitor (IBMX, 100µM) was applied, as well as PDE3 (Cilostamide, 1µM) and PDE4 (Ro 20-1724, 10µM) specific inhibitors. Results A desensitization to β-adrenergic receptor stimulation in AF myocytes was exclusively found in the cytosol, while no difference was seen in neither the RyR2 nor LTCC compartment in AF versus Ctl. Similar effects were observed upon 5-HT stimulation with a significant desensitization in the cytosol, and no difference in the RyR2 compartment. On the contrary, AF myocytes displayed a significantly higher increase in cAMP levels compared to Ctl myocytes in the cytosol upon A2ARs stimulation. Importantly, no effect on cAMP levels was observed in the LTCC compartment after A2ARs or 5-HT stimulation. However, PDE3 inhibition on top of 5-HT stimulation showed a significantly smaller effect on cAMP levels in AF myocytes within the LTCC compartment. Conclusions Collectively, our data show that cAMP levels are highly compartmentalized in human atrial myocytes and differentially regulated by different GPCRs. Furthermore, PDEs are to a certain extent responsible for the compartmentalized effects of the different GPCRs. </jats:sec

P2869Role of PDE8 in cAMP dynamics in human atrial fibrillation

Abstract Background Cardiac arrhythmias, such as atrial fibrillation (AF), are often related to remodeling of membrane receptors and alterations in cAMP-dependent regulation of Ca2+ handling mechanisms. For instance, decreased L-type calcium current (ICa,L) density but upregulated RyR2 are major hallmarks of AF. These inhomogeneous AF-associated changes of protein phosphorylation point to a local regulation of PKA activity within these intracellular compartments. Local cAMP compartmentation and the role of phosphodiesterase (PDEs) have ben extensively studied in ventricular myocytes from animals. However, only a few studies have evaluated the contribution of PDEs to the pathophysiology of AF and the reason for the persistent AF-associated hypophosphorylation of the L-type calcium channel (LTCC) is currently unknown. The aim of this study was to investigate whether a change in the expression level of PDE8 in human atrium may affects cAMP nearby LTCC promoting the reduction of the ICa,L observed in persistent AF. Methods Atrial myocytes were isolated from tissue of 47 patients in sinus rhythm (SR) and with AF. Cells were then transfect with an adenovirus (Epac1-camps or pm-Epac1-camps) in order to express the (cytosolic or membrane, respectively) FRET-based cAMP sensor and cultured during 48 hours. Föster-resonance energy transfer (FRET) was used to measure cAMP in 232 isolated human atrial myocytes. Ro-20-1724 (10 μM), Cilostamide (1 μM) and PF-04957325 (30 nM) and IBMX (100 μM) were used as PDE4, PDE3, PDE8 and non-selective phosphodiesterases (PDEs) inhibitor respectively. Results Effects of PDE4 and especially PDE3 inhibition on cytosolic [cAMP] are reduced in AF. Pharmacological PDE8 inhibition induces only a small increase in basal intracellular [cAMP] in AF but it showed a big synergic effect when PDE4 was inhibit at the same time. By contrast, PDE8 inhibition dramatically increased basal [cAMP] in the subsarcolemmal compartment in AF while PDE3 or PDE4 inhibition had a smaller effect that didn't change between SR and AF. Conclusions PDE8 controls basal cytosolic cAMP levels in human atrial myocytes from patients with persistent AF while PDE3 effects tends to be reduced in these patients. Furthermore, PDE8 is the main PDE in controlling cAMP levels at the membrane in persistent AF. Thus, our study may provide a clue for the reported reduction of the ICa,L in persistent AF. </jats:sec

P5992Why aortic valve disease may persist after surgery? A joint basic science - Clinical effort

Abstract Background Diseases of the aortic valve are a common reason for heart surgery. Aortic stenosis (AS) is associated with pressure and aortic regurgitation (AR) with a volume overload of the left ventricle (LV). Over time both pathologies lead to systolic and diastolic heart failure, while progressive downregulation of β-adrenoceptors occurs. While LV re-remodeling occurs in the majority of patients after aortic valve surgery, LV dysfunction persists in one fourth of such patients and leads to a terminal heart failure. We aimed to investigate whether differential remodeling in the protein kinase A (PKA) dependent inotropic response in myocytes and myocardial tissue obtained from patients undergoing aortic valve surgery is associated with the LV re-remodeling after surgery. Methods Preoperatively, pro BNP levels were measured and left ventricular strain analysis via echocardiography was performed. Interventricular septal biopsy was obtained intraoperatively in 10 patients who underwent aortic valve surgery. In-vitro contractility was analyzed in myocardial tissue paced with 4 Hz at 37 °C. Freshly isolated cells were transduced with an adenovirus expressing a cytosolic Förster resonance energy transfer (FRET) based cAMP biosensor (Epac1-camps). After 48 hours of culture, Föster-resonance energy transfer (FRET) was used for the first time to measure cAMP in 60 isolated human ventricular myocytes. Isoprenaline (10 nM – 10 μM) was used for β-adrenoceptor activation and forskolin (10 μmol) to activate adenylyl cyclase directly. Results We found a significantly downregulated β-adrenergic sensitivity in cardiomyocytes of patients with aortic valve disease, although contractile response to forskolin was maintained. Furthermore, we found a clear association between reduced sensitivity to isoprenaline (i.e., high EC50 values) and low maximum effect size to isoprenaline in myocardial tissue of patients with aortic valve disease, pointing out relevant β-adrenoceptor dysfunction. There were no significant differences in basal myocardial force between tissue samples of patients with AR and AS. Conclusion Collectively, our data show a profound remodelling in the cAMP/PKA pathway in patients with aortic valve disease. These disturbances may have an impact on the postoperative ventricular function and possibly on the long-term LV re-remodelling after aortic valve surgery. </jats:sec