Novel Potential Interacting Partners of Fibronectin in Spontaneous Animal Model of Interstitial Cystitis

Feline idiopathic cystitis (FIC) is the only spontaneous animal model for human interstitial cystitis (IC), as both possess a distinctive chronical and relapsing character. Underlying pathomechanisms of both diseases are not clearly established yet. We recently detected increased urine fibronectin levels in FIC cases. The purpose of this study was to gain further insight into the pathogenesis by assessing interacting partners of fibronectin in urine of FIC affected cats. Several candidate proteins were identified via immunoprecipitation and mass spectrometry. Considerable changes in FIC conditions compared to physiological expression of co-purified proteins were detected by Western blot and immunohistochemistry. Compared to controls, complement C4a and thioredoxin were present in higher levels in urine of FIC patients whereas loss of signal intensity was detected in FIC affected tissue. Galectin-7 was exclusively detected in urine of FIC cats, pointing to an important role of this molecule in FIC pathogenesis. Moderate physiological signal intensity of galectin-7 in transitional epithelium shifted to distinct expression in transitional epithelium under pathophysiological conditions. I-FABP expression was reduced in urine and urinary bladder tissue of FIC cats. Additionally, transduction molecules of thioredoxin, NF-κB p65 and p38 MAPK, were examined. In FIC affected tissue, colocalization of thioredoxin and NF-κB p65 could be demonstrated compared to absent coexpression of thioredoxin and p38 MAPK. These considerable changes in expression level and pattern point to an important role for co-purified proteins of fibronectin and thioredoxin-regulated signal transduction pathways in FIC pathogenesis. These results could provide a promising starting point for novel therapeutic approaches in the future.     

The contribution of sensory inputs to the pattern generation of breathing

Current concepts of the basic neural control system and its modulation by afferent inputs are reviewed. It is emphasized that, in analogy with locomotion, the central pattern generator (CPG) for automatic metabolic respiration does not depend on any afferent feedback from receptors sensitive to the movements of the "pump," or the streams of pumped air, for its production of a rhythmic motor output provides the CPG receives some "drive" inputs above threshold and adequate bias. The operation for a variety of reflexes and feedback loops is of fundamental importance, however, for adapting the breathing pattern to the varying requirements for gas exchange to the many behavioural, nonmetabolic demands on the breathing apparatus which are competing with its primary metabolic control functions. The presentation is focussed also on available evidence that the respiratory CPG exerts powerful modulations on the transmission in these reflex pathways controlling the pattern of breathing and adjusting it to the various metabolic and behavioural demands. Mechanisms for "gating," "phasic gain changes," and "phase-dependent reflex reversal" are exemplified.     

A model of the central and reflex inhibition of inspiration in the cat

An attempt is made to summarize the results obtained in previous work from this and other laboratories on the steady state and transient relationships between the mechanical and neural events in breathing and their precise timing in the breathing cycle at different blood chemical demands for ventilation and at different body temperatures, and to fit these results into a functional and realistic model of the bulbo-pontine inspiratory off-switch mechanisms. The experimentally based requirements for the model are briefly described and listed. After a presentation of the model in qualitative terms its functional properties are considered quantitatively and compared with the performance of the real, biological system. This has been achieved by assuming some simple mathematical approximations for the activities of the introduced physiological parameters and their chemical “drive” dependence. The gaps in our present knowledge are pointed out and some key experiments suggested. The proposed model is consistent with the main conclusions reached in previous work from this laboratory that there are three neural submechanisms which are mainly responsible for the effects of increased CO₂ on ventilation: 1) a rise in the inspiratory off-switch threshold, 2) an increased rate of rise of the centrally generated inspiratory activity that projects to the off-switch mechanism (and to the spinal respiratory motoneurons), and 3) the vagal volume feed-back. Of these 1) and 2) are mainly responsible for the increase in tidal volume, whereas the vagal volume feed-back is mainly responsible for the increase in respiratory rate. The comparison between the model behaviour and experimental data suggest that the slight CO₂ sensitivity of the pulmonary stretch receptors recently reported on, has to be taken into account. The model studies have suggested the increase in respiratory rate with increased temperature may be due either to an increased rate of rise of inspiratory activity or to a decreased off-switch threshold, or both in combination. The mechanism controlling the expiratory durations are briefly discussed.     

Spotting altruistic dictator game players and mingling with them: the elective assortation of classmates

Altruism can evolve through assortation if the selfish advantage of egoistic individuals is outcompeted by the benefits of mutual cooperation between altruists. This selection process is possible if (a) individuals can distinguish altruists from egoists and (b) altruists cooperate electively with other altruists, leaving egoists no chance but to mingle with each other. This study investigates whether these two conditions are fulfilled in a natural setting. One hundred twenty-two students of six secondary school classes (age 10 to 19 years) played an anonymous dictator game, which functioned as a measure of altruism. Afterwards and unannounced, the students had to estimate their classmates' decisions and did so better than chance. Sociometry revealed that the accuracy of predictions depended on social closeness. Friends and disliked classmates were judged more accurately than liked classmates or those met with indifference. Moreover, altruists were friends with more altruistic persons than were egoists. The results confirm the existence of the two prerequisites for the evolution of altruism through assortation: the predictability of altruistic behavior and the association of altruists.     

A Combined Laser Microdissection and Mass Spectrometry Approach Reveals New Disease Relevant Proteins Accumulating in Aggregates of Filaminopathy Patients

Filaminopathy is a subtype of myofibrillar myopathy caused by mutations in FLNC, the gene encoding filamin C, and histologically characterized by pathologic accumulation of several proteins within skeletal muscle fibers. With the aim to get new insights in aggregate composition, we collected aggregates and control tissue from skeletal muscle biopsies of six myofibrillar myopathy patients harboring three different FLNC mutations by laser microdissection and analyzed the samples by a label-free mass spectrometry approach. A total of 390 proteins were identified, and 31 of those showed significantly higher spectral indices in aggregates compared with patient controls with a ratio >1.8. These proteins included filamin C, other known myofibrillar myopathy associated proteins, and a striking number of filamin C binding partners. Across the patients the patterns were extremely homogeneous. Xin actin-binding repeat containing protein 2, heat shock protein 27, nebulin-related-anchoring protein, and Rab35 could be verified as new filaminopathy biomarker candidates. In addition, further experiments identified heat shock protein 27 and Xin actin-binding repeat containing protein 2 as novel filamin C interaction partners and we could show that Xin actin-binding repeat containing protein 2 and the known interaction partner Xin actin-binding repeat containing protein 1 simultaneously associate with filamin C. Ten proteins showed significant lower spectral indices in aggregate samples compared with patient controls (ratio <0.56) including M-band proteins myomesin-1 and myomesin-2. Proteomic findings were consistent with previous and novel immunolocalization data. Our findings suggest that aggregates in filaminopathy have a largely organized structure of proteins also interacting under physiological conditions. Different filamin C mutations seem to lead to almost identical aggregate compositions. The finding that filamin C was detected as highly abundant protein in aggregates in filaminopathy indicates that our proteomic approach may be suitable to identify new candidate genes among the many MFM patients with so far unknown mutation.Myofibrillar myopathies (MFM)¹ encompass a genetic and clinically heterogenous group of muscle disorders characterized by focal myofibrillar destruction and massive protein aggregation within skeletal muscle fibers (1). The mechanisms leading to aggregate formation are not well understood but an impairment of protein degradation systems seems to play an important role (2). Immunohistochemical studies identified various proteins accumulating in these aggregates (3) but the precise composition is unknown so far. The latter may give us important new insights in the pathogenesis of MFM.Filaminopathy is a subtype of MFM caused by mutations in the rod domain of FLNC, the gene encoding FLNc (4–7). Filamins are a small group of large cytoskeletal proteins that crosslink F-actin filaments and act as scaffold for transmembrane receptors, signaling, and adapter proteins. FLNc is the striated muscle-specific isoform that cross-links actin at the Z-disc level and is important for the maintenance of myofibrillar integrity. Filaminopathy is leading to a progressive muscle weakness usually manifesting between the fourth and sixth decade of life. The pattern of severely and hardly affected muscles detected by magnetic resonance imaging is very homogeneous across patients with different FLNC mutations (8, 9, and unpublished data). In advanced stages of the disease, patients generally lose the ability to walk and show respiratory insufficiency because of weakness of respiratory muscles. A cardiac involvement is also frequent (4, 5).Over the last years proteomics has developed to a promising tool for the analysis of the skeletal muscle proteome. Several studies including gel-based and mass spectrometric approaches have been performed that mainly aimed at the global cataloguing and biochemical characterization of the whole rodent muscle proteome or of cellular substructures under physiological and aging conditions (10,11, and for review see (12)). In aged muscles, for example, abundance changes have been detected for proteins involved in metabolism, contractile activity, myofibrillar remodeling, and stress response (12). In biomedical research global studies focused on the identification of novel panels of protein biomarker candidates for neuromuscular diseases: Analyses of muscle tissue of the dystrophin deficient mouse model of Duchenne muscular dystrophy (mdx mouse) identified altered levels of protein biomarkers involved in nucleotide metabolism, cellular stress response, energy metabolism, and ion handling (for review see (13)). Significant alterations of proteins playing an important role in various metabolic pathways were found in a mouse model for hypokalemic myopathy (14). In a 2-DE study of total muscle extracts from dysferlinopathy patients, 35 proteins were found to be differentially expressed (15). Metabolic and contractile proteins represented the majority of the changes suggesting an active process of muscle regeneration and a remodeling of fiber type as a result of dysferlin deficiency. In sporadic inclusion body myositis (sIBM), proteins associated with amyloidosis were up-regulated (16). A comparison of protein expression in sIBM to non-IBM inflammatory myopathies indicated an impairment of detoxification, energy metabolism, and protein folding in sIBM (16).All of the above mentioned studies used total muscle protein extracts or soluble cytosolic protein fractions resulting in limitations because of high sample heterogeneity and complexity. Muscle tissue is composed of a complex mixture of different cell types from epi-, endo-, perimysium, muscle spindles, blood vessels etc. and changes in the protein abundance might be the result of differences in sample composition rather than disease-related effects. Additionally, the presence of a number of highly abundant and unusually large proteins (e.g. actin, myosin, titin) hampers the detection of potentially relevant low abundant disease-associated proteins. Those limitations could be bypassed by application of technologies allowing specific isolation of physiologically and pathophysiologically relevant muscular substructures before proteomic analysis, such as (sub) cellular microdissection. This can either be performed manually (17, 18) or laser-assisted, and provides unparalleled accuracy obtaining pure cell populations or even pure subcellular structures (for review see (19)). Laser microdissection (LMD) in combination with mass spectrometry has been successfully applied for reducing body myopathy (RBM) (20), a hereditary muscle disease histologically characterized by intracytoplasmic inclusions, so called reducing bodies. FHL1 (four and a half LIM domain 1) was identified as the most prominent component of isolated reducing bodies. Subsequently performed mutational analysis revealed different pathogenic FHL1 mutations in RBM patients. This was the first example of an essential contribution of a proteomic approach to the identification of the disease-causing mutation in a hereditary myopathy.The aim of our study was to identify proteins that accumulate in protein aggregates within muscle fibers in MFM associated with FLNC mutations in order to get new insights in the pathomechanisms and to detect specific protein biomarker candidates for differential diagnosis. Skeletal muscle biopsies of six patients with three different FLNC mutations have been included in this study. LMD in combination with a newly established label-free mass spectrometry approach reproducibly identified and relatively quantified 390 proteins in total. We were able to detect new components of protein aggregates in filaminopathy and describe and characterize the so far unknown interaction between FLNc and Xin actin-binding repeat containing protein 2 (Xirp2).     

SMAD‐proteins as a molecular switch from hypertrophy to apoptosis induction in adult ventricular cardiomyocytes

Heart failure development goes along with a transition from hypertrophic growth to apoptosis induction. In adult cardiomyocytes SMAD proteins are only activated under apoptotic, but not under hypertrophic conditions and are increased at the transition to heart failure. Therefore, SMADs could be candidates that turn the balance from hypertrophic growth to apoptosis resulting in heart failure development. To test this hypothesis we infected isolated rat ventricular cardiomyocytes with adenovirus encoding SMAD4 (AdSMAD4) and investigated the impact of SMAD4 overexpression on the development of apoptosis and hypertrophy under stimulation with phenylephrine (PE). Infection of cardiomyocytes with AdSMAD4 significantly enhanced SMAD‐binding activity while apoptosis after 24 and 36 h infection did not rise. But when SMAD4 overexpressing cardiomyocytes were incubated with PE (10 µM), the number of apoptotic cells increased (Ctrl: 94.97 ± 6.91%; PE: 102.48 ± 4.78% vs. AdSMAD4 + PE: 118.64 ± 3.28%). Furthermore expression of caspase 3 as well as bax/bcl2 ratio increased in SMAD4 overexpressing, PE‐stimulated cardiomyocytes. In addition, the effects of SMAD4 overexpression on PE‐induced hypertrophic growth were analyzed. Protein synthesis 36 h after AdSMAD4 infection was comparable to control cells, whereas the increase in protein synthesis stimulated by phyenylephrine was significantly reduced in SMAD4 overexpressing cells (134.28 ± 10.02% vs. 100.57 ± 8.86%). SMAD4 triggers the transition from hypertrophy to apoptosis in ventricular cardiomyocytes. Since SMADs are increased under several pathophysiological conditions in the heart, it can be assumed that it triggers apoptosis induction and therefore contributes to negative remodeling and heart failure progression. J. Cell. Physiol. 220: 515–523, 2009. © 2009 Wiley‐Liss, Inc.