Η μυο-ειδική πρωτεΐνη δεσμίνη, αποτελεί μέλος των πρωτεϊνών του κυτταροσκελετού των ενδιαμέσων ινιδίων και εκφράζεται στους λείους και τους γραμμωτούς μυς. Στους συσταλτούς μύες, το πλέγμα του κυτταροσκελετού της Δεσμίνης περιβάλλει τους Ζ-δίσκους διασυνδέοντάς τους, ενώ παράλληλα συνδέει μεταξύ τους τις συσταλτές περιοχές της μυικής ίνας με την σαρκοπλασματική μεμβράνη, με διάφορα οργανίδια και με τον πυρήνα. Για να προσδιορίσουμε τους ακριβείς μηχανισμούς δράσης της δεσμίνης χρησιμοποιήσαμε το σύστημα υβριδισμού των ζυμών – yeast two hybrid screen system – προκειμένου να ανιχνεύσουμε πρωτεΐνες που αλληλεπιδρούν με τη δεσμίνη. Χρησιμοποιήσαμε ως «δόλωμα» αλληλουχίες των άκρων του μορίου της δεσμίνης του αμινο-τελικού και το καρβόξυ-τελικού. Μελετώντας τις πρωτεΐνες που προέκυψαν, διαπιστώσαμε ότι το αμινο-τελικό άκρο της δεσμίνης αλληλεπιδρά με διάφορες μιτοχονδριακές πρωτεΐνες. Με το ίδιο σύστημα αποκαλύψαμε αλληλεπιδράσεις της δεσμίνης με λυοσωματικές πρωτεΐνες όπως η καθεψίνη D και η προσαποσίνη οι οποίες αλληλεπιδρούν με το αμινοτελικό άκρο της δεσμίνης. Η καθεψίνη D είναι μια λυοσωματική πρωτεάση, που οδηγείται και ωριμάζει πλήρως στα λυοσώματα ενώ η προσαποσίνη είναι ένα πρόδρομο λυοσωματικό μόριο με πρωτεόλυση του οποίου, εντός του λυοσώματος, προκύπτουν οι σαποσίνες Α έως D. Η καθεψίνη D αποτελεί δείκτη καταστάσεων αυτοφαγία και τελευταία φαίνεται ότι επεμβαίνει σε φαινόμενα απόπτωσης επάγωντάς την κατά περίπτωση. Η αλληλεπίδραση της δεσμίνης με την καθεψίνη D επιβεβαιώθηκε και με βιοχημικές τεχνικές (in vitro) όπως η συνεργιστική ανοσοκαθίζηση /ανοσοκατακρήμνιση (co-immuno-precipitation) και η τεχνική GST pull-down. Μετά και από αυτές τις in vitro αποδείξεις, φαίνεται πως μάλλον συμβαίνει ευθεία αλληλεπίδραση μεταξύ της δεσμίνης και της καθεψίνης D. Γι’ αυτό, και με βάση όσα είναι γνωστά για την καθεψίνη D, προτείνουμε μια νέα λειτουργία του κυτταροσκελετού της δεσμίνης πιθανόν στην μετακίνησης και τη δημιουργία των λυοσωμάτων αλλά και έναν νέο ρυθμιστικό ίσως ρόλο της, σε διαδικασίες αυτοφαγίας και απόπτωσης, μέσω της πρόσδεσής της με σημαντικά μόρια ρυθμιστές τέτοιων διαδικασιών.................... / Desmin is the muscle - specific member of the intermediate filament family of cytoskeletal proteins, expressed both in striated and smooth muscle tissues. In mature striated muscle fibers, the desmin filament lattice surrounds the Z-discs, interconnects them to each other and links the entire contractile apparatus to the sarcolemmal cytoskeleton, cytoplasmic organelles and the nucleus. In order to identify the exact mechanisms of desmin’s action, we performed a yeast two-hybrid screen for desmin-interacting proteins. For this purpose, we used as baits the two non helical terminal regions of the desmin molecule, the amino (head)- and the carboxy (tail)- terminal domain. We have found that the head domain of desmin potentially interacts with two new groups of proteins, mitochondria and lysosome related. Specifically, in the second category, we have revealed an association of the head domain of desmin with Cathepsin D (one of the lysosomal proteinases) and prosaposin (a single precursor which gives rise to Saposins A-D by proteolytic cleavage in lysosomes and is also referred to as sphigolipid activator proteins). In addition to its targeting to lysosomes, Cathepsin D is also involved in apoptosis and autophagy processes. This protein interaction result has been retested. The interaction between cathepsin D and desmin has also been further confirmed both with reverse yeast transformation as well as biochemical assays such as co-immunoprecipitation and GST pull down assay. The above described strong evidence of direct interaction between desmin and cathepsin D, has allowed us to propose a novel function of desmin IFs in lysosomal trafficking and/or as a new regulator of autophagy and apoptotic cell death.

Background: Snoring and obstructive sleep apnea (OSA) are both common disorders with a number of negative health effects. The safety and efficacy of treating snoring and OSA surgically have been questioned and there has been a lack of studies in the field. Aims: 1) To investigate the frequency of serious complications, including death, after surgery for the treatment of snoring and sleep apnea; 2) to evaluate the effect on daytime sleepiness after radiofrequency surgery of the soft palate in snoring men with mild or no OSA; 3) to evaluate the effect of tonsillectomy on sleep apnea in adults with OSA and tonsillar hypertrophy; 4) to investigate the morphology and cytoarchitecture of muscle fibers in human soft palatal muscles with immunohistochemical and morphological techniques. Methods and results: In paper 1, a retrospective database study. All Swedish adults who were treated surgically because of snoring or OSA from January 1997 to December 2005 were identified in the National Patient Register. None of the surgically treated patients died in the peri- and postoperative period. Severe complications were recorded in 37.1 of 1,000 patients treated with uvulopalatopharyngoplasty (UPPP), in 5.6 of 1,000 patients after uvulopalatoplasty (UPP) and in 8.8 of 1,000 patients after nasal surgery. In paper 2, the study was designed as a randomized, controlled trial. 35 snoring men with mild or no OSA were randomized to either radiofrequency or sham surgery of the soft palate. Radiofrequency surgery was not found to be effective since there was no significant difference between the two groups in relation to the Epworth Sleepiness Scale (ESS) or apnea-hypopnea index (AHI) at follow-up. Paper 3 was a prospective study, including 28 patients with an AHI of >10 and with large tonsils. In these patients, tonsillectomy was an effective treatment for OSA; the mean AHI was reduced from 40 units/h to 7 units/h (p<0.001), and the mean ESS was reduced from 10.1 to 6.0 (p<0.001) at the six-month follow-up after surgery. Minor and moderate swallowing dysfunction was found in seven of eight patients investigated before surgery and the swallowing function improved in 5 of them after surgery, while no one deteriorated. In paper 4, we investigated the morphology and cytoarchitecture in normal soft palate muscles. Human limb muscles were used as reference. The findings showed that the soft palate muscle fibers have a cytoskeletal architecture and cellmembrane complex that differs from that of the limb muscles. Conclusions No case of death related to surgery was found among 4,876 patients treated with UPPP, UPP or nasal surgery for snoring or OSA in Sweden between 1997 and 2005. Radiofrequency surgery of the soft palate has no effect on daytime sleepiness, snoring or apnea frequency in snoring men with mild or no OSA. Tonsillectomy can be an effective treatment for OSA in adults with large tonsils. A subgroup of muscle fibers in the human soft palate appears to have special biomechanical properties and their unique cytoarchitecture must be taken into account while assessing function and pathology in oropharyngeal muscles. / Snarkning och obstruktiv sömnapné (OSA) är idag en global folksjukdom. Snarkning är det ”oljud” som uppstår när luftvägen under sömn förminskas och vävnaden börjar vibrera under andning. Vid obstruktiv sömnapné faller vävnaden samman och blockerar luftflödet till lungorna. Ett andningsuppehåll, en s.k. apné inträffar. Ett andningsuppehåll kan pågå allt ifrån några sekunder till mer än en minut och kan uppstå hundratals gånger per natt. För att klassificeras som en patologisk apné enligt internationell standard måste andningsuppehållet vara längre än 10 sek. Snarksjukdomen förvärras sannolikt över tid och övergår succesivt i obstruktiv sömnapné med ökande antal andningsuppehåll under sömn. Detta leder till ett stresspåslag för kroppen med oftast uttalad dagtrötthet och en mängd negativa hälsoeffekter. Snarksjukdom och sömnapné ökar risken för bl.a. högt blodtryck och hjärt-kärlsjukdom samt också för att den drabbade ska orsaka trafikolyckor på grund av försämrad koncentrationsförmåga och trötthet. En del av den negativa utvecklingen från snarkning till sömnapné anses bero på att snarkvibrationer kan ge neuromuskulära skador i gom och svalg. Dessa vävnadsskador anses också vara orsaken till att personer som snarkat länge ofta uppvisar störd sväljningsfunktion i form av felsväljning, där maten i uttalade fall hamnar i luftstrupen istället för i matstrupen. I dagsläget är förstahandsbehandling vid sömnapné CPAP, en mask som placeras över näsa och mun och som skapar ett övertryck i luftvägen vilket förhindrar att luftvägen faller samman och att andningsstopp uppstår. CPAP har enligt flera studier den bästa effekten mot andningsuppehåll. En annan vanlig behandling är en bettskena som för underkäken nedåt och framåt så att luftvägen bli mer öppen. Bettskenan är en vanlig och effektiv behandlingsmetod för personer utan kraftig övervikt vid vanemässig snarkning eller måttlig sömnapné. För ett tjugotal år sedan var kirurgi förstahandsmetoden vid behandling av snarkning och måttlig sömnapné. Man utförde då ofta operationer i svalg och gomm, s.k. gomplastiker. Bruket av kirurgisk behandling har dock minskat med tiden, dels p.g.a. biverkningar men också för att det saknades vetenskapliga studier som bevisade att kirurgin gav önskad och långsiktig effekt. Kirurgi utgör dock fortfarande ett komplement till behandling av snarkning och sömnapné när CPAP eller bettskena av olika skäl inte fungerar eller kan tolereras av patienten. 8 Även barn kan lida av snarkning och sömnapné men behandlingsprinciperna för barn skiljer sig från dem hos vuxna och berörs inte i avhandlingen. I denna avhandling studeras: i) biverkningsfrekvenser efter olika typer av snarkkirurgi, ii) effekten av radiovågsbehandling i mjuka gommen på vuxna män med snarkning, iii) effekten av att operera bort halsmandlarna på vuxna med sömnapné och stora halsmandlar, iv) muskelvävnadens struktur och molekylära uppbyggnad i mjuka gommen hos friska personer som inte snarkar. Avhandlingen består av fyra delstudier: 1. En registerstudie med kartläggning av svåra biverkningar efter kirurgi i form av uvulopalatopharyngoplastik, uvulupalatoplastik samt näskirurgi för behandling av sömnapné och snarkning och utfört i Sverige mellan åren 1997-2005. Studien omfattade 4 876 patienter. Inga dödsfall noterades. Komplikationsrisken var störst vid operationer där man tog bort delar av mjuka gommen samt halsmandlarna, där i snitt 37 av 1000 opererade fick biverkningar, framförallt p.g.a. infektioner eller blödningar. 2. I en prospektiv, randomiserad placebostudie utvärderades effekten av radiovågsbehandling i mjuka gommen vid snarkning och lindrig sömnapne. Trettiotvå patienter lottades till att få radiovågsbehandling eller placebo behandling. Patienterna visste inte vilken grupp de tillhörde. Vid uppföljning efter 12 månader var det inga statistiska belägg för att radiovågsbehandling minskade vare sig antal andningsuppehåll eller dagtrötthet. 3. Effekten av att ta bort halsmandlarna på patienter med stora halsmandlar och olika grad av sömnapné utvärderades i denna studie. Totalt deltog 28 patienter. Vid uppföljning 6 månader efter operationen hade antalet andningsuppehåll sjunkit drastiskt, från i snitt 40 till 7 andningsuppehåll per timme nattsömn. Inga allvarliga biverkningar uppstod. Dessa fynd talar för att man som förstahandsmetod ska erbjuda patienter med sömnapné och stora halsmandlar att ta bort halsmandlarna. 4. I detta projekt undersökte vi utseendet och uppbyggnaden av cellskelettet i två normala muskler i mjuka gommen hos friska personer utan känd snarkning och sömnapné. Muskler från armar och ben användes som referens. Fynden i studien visar att de normala muskelfibrernas uppbyggnad i mjuka gomen skiljer sig från jämförade muskler i armar och ben. Detta kan vara ett uttryck för en evolutionär utveckling för att möjligöra de komplexa funktioner som krävs av svalgets muskulatur. 9 Sammanfattningsvis kan vi konstatera: Att inga dödsfall har skett i Sverige efter operationer i gom, svalg eller näsa, utförda för att behandla snarkning och sömnapné under åren 1997 till 2005. Att radiovågsbehandling av mjuka gommen hos snarkande män med lindrig sömnapné inte har någon effekt på dagtrötthet, snarkning eller andningsuppehåll vid uppföljning efter 12 månader. Metoden kan därför inte rekommenderas. Att när man opererar bort stora halsmandlar på personer med andningsuppehåll så leder detta ofta till att andningsuppehållen minskar drastiskt. Metoden kan därför oftast rekommenderas som en förstahandsbehandling för denna patientgrupp. Att mjuka gommens muskelfibrer är uppbyggda på ett unikt sätt indikerar att deras specifika biomekaniska egenskaper skiljer sig från referens muskler i armar och ben.

Πρόσφατες μελέτες παρέχουν ενδείξεις για τη συμμετοχή του οξειδωτικού στρες στην ανάπτυξη καρδιαγγειακών νοσημάτων. Το οξειδωτικό στρες έχει συσχετιστεί ισχυρά με τον κυτταρικό θάνατο και διαδικασίες καρδιακής αναδόμησης, που αποτελούν χαρακτηριστικά της καρδιακής ανεπάρκειας. Μύες χωρίς δεσμίνη, σημαντική πρωτεΐνη των μυϊκών ενδιαμέσων ινιδίων, αναπτύσσουν διατατική μυοκαρδιοπάθεια και καρδιακή ανεπάρκεια, η οποία χαρακτηρίζεται από μιτοχονδριακές ανωμαλίες και κυτταρικό θάνατο μαζί με εκτεταμένες εναποθέσεις ασβεστίου και ίνωση, προσφέροντας έτσι ένα πολύ καλό μοντέλο καρδιακής ανεπάρκειας.Διάφορες κυτταρικές και βιοχημικές αλλοιώσεις στην καρδιά των μυών αυτών υποδηλώνουν έντονα ότι το οξειδωτικό στρες είναι ένας σημαντικός μηχανισμός που συμβάλλει στην παθογένεση αυτού του φαινότυπου. Οι ανωμαλίες στη μιτοχονδριακή δομή και λειτουργία, οι οποίες χαρακτηρίζουν το φαινότυπο του μυός χωρίς δεσμίνη, προσφέρουν τις πιο σημαντικές ενδείξεις για την ύπαρξη οξειδωτικού στρες, καθώς η αναπνευστική αλυσίδα είναι η πιο σημαντική πηγή δραστικών Ενώσεων Οξυγόνου (ΔΕΟ ή Reactive Oxygen Species - ROS) στα μυοκαρδιοκύτταρα. Προκειμένου να διασαφηνιστεί η ύπαρξη οξειδωτικού στρες στο μυοκάρδιο απουσία δεσμίνης και συνεπώς η συμμετοχή του στην εξέλιξη του μυοεκφυλισμού, επιχειρήθηκαν τόσο in vitro, όσο και in vivo προσεγγίσεις. Η ύπαρξη οξειδωτικής καταπόνησης διερευνήθηκε σε πρωτογενείς καλλιέργειες ενήλικων μυοκαρδιοκυττάρων. Επιχειρήθηκε η ενίσχυση του αντιοξειδωτικού αμυντικού συστήματος, έτσι ώστε να αποτιμηθεί, τόσο η συμβολή του οξειδωτικού στρες στο μυοεκφυλισμό, όσο και η πιθανή θεραπευτική δράση των αντιοξειδωτικών στρατηγικών. Προκειμένου να αποτιμηθούν τα επίπεδα ενδοκυτταρικής οξειδωτικής καταπόνησης αναπτύχθηκε νέα μέθοδος απομόνωσης ενήλικων μυοκαρδιοκυττάρων από μυ. Επειδή τα μιτοχόνδρια αποτελούν το κύριο στόχο των παρατηρούμενων αλλοιώσεων, επιχειρήθηκε πρώτα ο προσδιορισμός των γενικών αλλαγών που παρατηρούνται στο μιτοχονδριακό πρωτέωμα. Πράγματι, οι παρατηρούμενες αλλαγές στα επίπεδα πρωτεϊνικής έκφρασης ενίσχυσαν την αρχική υπόθεση. Στη συνέχεια εκτιμήθηκαν τα ενδοκυτταρικά επίπεδα ROS σε καλλιέργειες ενήλικων 8 μυοκαρδιοκυττάρων χρησιμοποιώντας φθορίζοντες ιχνηθέτες. Ανάλυση των αποτελεσμάτων έδειξε ότι υπάρχουν αυξημένα επίπεδα ROS στα μυοκαρδιοκύτταρα απουσία δεσμίνης. Επιπλέον, διερευνήθηκε το μιτοχονδριακό μεμβρανικό δυναμικό, το οποίο είναι ενδεικτικό της σωστής μιτοχονδριακής λειτουργίας, χρησιμοποιώντας ειδικό φθορίζοντα ιχνηθέτη. Διαπιστώθηκε ότι υπάρχουν σημαντικές αλλοιώσεις σε αρκετά μυοκαρδιοκύτταρα απουσία δεσμίνης. Με σκοπό (1) να επιβεβαιωθεί η ύπαρξη οξειδωτικού στρες in vivo, (2) να αποτιμηθεί η συμβολή του στο φαινότυπο του μυός χωρίς δεσμίνη και (3) να εκτιμηθεί η θεραπευτική δυνατότητα της προστασίας έναντί του, το αντιοξειδωτικό αμυντικό σύστημα ενισχύθηκε in vivo, χρησιμοποιώντας το μυ χωρίς δεσμίνη ως μοντέλο καρδιακής ανεπάρκειας. Για το σκοπό αυτό δημιουργήθηκαν διαγονιδιακοί μύες που υπερεκφράζουν στο μυοκάρδιο τα αντιοξειδωτικά ένζυμα καταλάση και υπεροξειδική δυσμουτάση (MnSOD). Η καταλάση αποτοξινώνει τα κύτταρα από το H2O2 μετατρέποντας το σε νερό και οξυγόνο. Η μυοκαρδιακή υπερέκφραση καταλάσης μελετήθηκε σε υπόβαθρο απουσίας δεσμίνης. Το επίπεδο υπερέκφρασης αποτιμήθηκε σε επίπεδο τόσο πρωτεϊνικό, όσο και ενζυμικής ενεργότητας. Ο καρδιοπροστατευτικός ρόλος της καταλάσης αποτιμήθηκε ως συνιστώσα των επιπέδων ινωδών αλλοιώσεων, της υπερδομής και της καρδιακής συστολικής λειτουργίας. Η υπερέκφραση καταλάσης στο μυοκάρδιο μυών χωρίς δεσμίνη οδηγεί σε σημαντική μείωση των ενδοκυτταρικών επιπέδων ROS και της έκτασης ινωδών αλλοιώσεων, μειώνει το μυοεκφυλισμό και βελτιώνει την καρδιακή συστολική λειτουργία. Τα αποτελέσματα αυτά επιβεβαιώνουν τη συμβολή του οξειδωτικού στρες και ειδικά του H2O2 στην ανάπτυξη μυοκαρδιοπάθειας και καρδιακής ανεπάρκειας στο μυ χωρίς δεσμίνη και υπογραμμίζουν τη θεραπευτική δυνατότητα της υπερέκφρασης καταλάσης. Η MnSOD εντοπίζεται στη μιτοχονδριακή μήτρα και μετατρέπει το υπεροξειδικό ανιόν σε υπεροξείδιο του υδρογόνου. Η καρδιακή υπερέκφραση MnSOD μελετήθηκε σε υπόβαθρο απουσίας δεσμίνης. Υπερέκφραση της MnSOD μόνο σε ενδιάμεσα επίπεδα οδηγεί σε μείωση των επιπέδων του υπεροξειδικού ανιόντος και των ινωδών αλλοιώσεων στο μυοκάρδιο απουσία δεσμίνης. Επιπλέον, παρατηρήθηκε βελτίωση της μυοκαρδιακής υπερδομής, καθώς και μέτρια βελτίωση της καρδιακής συστολικής λειτουργίας. Η υποβολή μυών χωρίς δεσμίνη που υπερεκφράζουν MnSOD σε υποχρεωτική άσκηση είχε ως αποτέλεσμα το θάνατο. Αυτή η κατάληξη δεν παρατηρήθηκε όταν στο μυοκάρδιο χωρίς δεσμίνη 9 υπερεκφράζονταν τόσο η καταλάση, όσο και η MnSOD. Αυτό το αποτέλεσμα υποδηλώνει ότι το H2O2 είναι σημαντικός διαμεσολαβητής της παρατηρούμενης θνησιμότητας. Είναι ενδιαφέρον ότι η MnSOD έχει «μεικτή» συμβολή στην αποτοξίνωση από ΕΜΟ, καθώς διασπά μία δραστική ένωση δημιουργώντας ταυτόχρονα μία άλλη. Είναι συνεπώς πολύ σημαντικό η υπερέκφραση αυτού του αντιοξειδωτικού ενζύμου να πραγματοποιείται με επίγνωση των επιβλαβών συνέπειών του. Συνολικά, τα αποτελέσματα που παρουσιάζονται εδώ επιβεβαιώνουν τη συμβολή της οξειδωτικής καταπόνησης στην ανάπτυξη κληρονομικής μυοκαρδιοπάθειας και καρδιακής ανεπάρκειας, καθώς και τη θεραπευτική ικανότητα των διαφορετικών αντιοξειδωτικών στρατηγικών και του συνδυασμού τους. / Recent studies support the contribution of oxidative stress in the development of cardiovascular diseases. Oxidative stress has been strongly linked to cell death and cardiac remodeling processes, all hallmarks of heart failure. Mice null for desmin, which is the major muscle specific intermediate filament protein, develop dilated cardiomyopathy and heart failure characterized by mitochondrial defects and cardiomyocyte death accompanied by extensive calcification and fibrosis, thus providing a very good model for heart failure. Several cellular and biochemical alterations in the hearts of these mice strongly suggested that oxidative stress is one of the mechanisms contributing to the pathogenesis of the phenotype. The defects in mitochondrial structure and function, hallmarks of the desmin null mouse phenotype, provide the most important indications for the existence of oxidative stress, as the respiratory chain is the most important source of reactive oxygen species (ROS) in cardiomyocytes. In order to delineate the existence of oxidative stress in the desmin null myocardium and therefore its participation in the development of the myocardial degeneration we sought both in vitro and in vivo approaches. The existence of oxidative stress was addressed in primary adult cardiomyocytes. The reinforcement of the antioxidant defense system was pursued, in order to assess the contribution of oxidative stress in the myocardial degeneration, as well as the therapeutic potential of antioxidant strategies. To assess intracellular oxidative stress a new method for the isolation of adult mouse cardiomyocytes was developed. Since mitochondria were the target of pathology, we wanted to first determine global changes in the mitochondrial proteome. The observed changes in protein levels reinforced the original hypothesis. Intracellular reactive oxygen species were measured using fluorescent probes in adult cardiomyocyte cultures. Analysis of the above data showed that there are increased levels of ROS in desmin null cardiomyocytes. Furthermore, the mitochondrial membrane potential, which is indicative of proper mitochondrial function, was investigated using a fluorescent probe. It was found altered in a subset of the desmin null cardiomyocytes. In order to (1) verify the existence of oxidative stress in vivo, (2) assess its contribution to the phenotype of desmin null mice and (3) evaluate the therapeutic 5 potential of protecting against it, the antioxidant defense system was fortified in vivo using the desmin null mouse as a heart failure model. Towards this goal transgenic mice overexpressing the antioxidant genes catalase and manganese superoxide dismutase (MnSOD) were created. Catalase detoxifies the cells from hydrogen peroxide by converting it to water and oxygen. Cardiac specific overexpression of catalase was brought to a desmin null background. The level of overexpression was assessed by measuring protein levels and enzyme activity. The cardioprotective effect of catalase was assessed in terms of fibrotic lesion extent, ultrastructure and cardiac systolic function. Overexpression of catalase in the heart of desmin null mice leads to marked decrease in intracellular ROS levels and significant decrease in fibrotic areas, ameliorates the myocardial degeneration and improves cardiac function. These data support the contribution of oxidative stress and in particular of the ROS hydrogen peroxide in the development of cardiomyopathy and heart failure in the desmin null mouse and underscore the therapeutic potential of catalase overexpression. MnSOD in localized in the mitochondrial matrix and converts superoxide anion to hydrogen peroxide. Cardiac specific overexpression of MnSOD was studied in a desmin null background. Overexpression of MnSOD only at moderate levels leads to a significant reduction of fibrotic lesion in the desmin null myocardium. Furthermore, an improvement of the myocardial ultrastructure was observed, as well as a moderate improvement of cardiac systolic function. These data suggest that another ROS, superoxide anion, contributes to the development of cardiomyopathy and heart failure in the desmin null mouse and that MnSOD, when overexpressed at moderate levels, offers cardioprotective effect. When the mice overexpressing MnSOD in the desmin null myocardium were challenged to exercise an absolute reduction of survival was observed. This defect was completely reversed when desmin null mice overexpressed both MnSOD and catalase. This suggests that hydrogen peroxide is an important mediator of the observed lethality. It is of note that MnSOD retains a contradictory antioxidant role, both breaking down and creating a specific ROS. It is therefore of paramount importance that this antioxidant enzyme is employed with caution and awareness of its deleterious effects. Overall, the data presented here demonstrate the contribution of oxidative stress in the development of inherited cardiomyopathy and heart failure, as well as the therapeutic potential of different antioxidant strategies, and their combination.

Os mecanismos responsáveis pela fibrose hepática na infância são pouco conhecidos. Crianças com atresia das vias biliares, quando submetidas a portoenterostomia a Kasai com sucesso, se tornam anictéricas mas mesmo assim desenvolvem cirrose a longo prazo. Da mesma forma, a ocorrência de estenoses biliares segmentares intra-hepáticas no pós-operatório de transplante hepático podem levar ao desenvolvimento de cirrose em todo o órgão. Tais fatos sugerem que mecanismos endócrinos ou parácrinos estejam envolvidos na fibrogênese hepática. Para elucidar este processo o modelo de ligadura seletiva das vias biliares em ratos jovens foi desenvolvido em nosso laboratório. Usando este modelo, identificamos mudanças na expressão do gene da alfa-actina de músculo liso, tanto no parênquima hepático obstruído como no parênquima hepático adjacente à obstrução. No entanto, o perfil de expressão gênica da desmina, uma proteína presente em níveis elevados durante a ativação das células estreladas hepáticas e o TGF-beta1, principal citocina pró-fibrogênica, não demostraram diferenças significantes quando analisados pelo método do RT-PCR semiquantitativo. Assim, os mecanismos moleculares envolvidos na modulação da fibrogênese hepática nesse modelo experimental não estão totalmente compreendidos. A metodologia do qRT-PCR (PCR em tempo real), têm sido previamente descrita como um método mais preciso e sensível, possibilitando a detecção do aumento do número de cópias do gene à medida que ocorre a amplificação, enquanto que o método do RT-PCR semiquantitativo a análise dos transcritos só é realizada após a etapa da amplificação. O objetivo deste estudo foi avaliar as alterações moleculares no modelo experimental de ligadura seletiva das vias biliares e comparar os resultados obtidos entre os métodos do RT-PCR semiquantitativo e do qRT-PCR em tempo real. Foi realizada a ligadura seletiva do ducto biliar em ratos Wistar com 21 dias de vida, os grupos foram separados de acordo com o momento da morte: 7 ou 60 dias após a cirurgia. A expressão da desmina, alfa actina de músculo liso e TGF-beta1 foi avaliada no tecido do parênquima hepático com obstrução biliar (ducto ligado - DL) e no parênquima hepático adjacente à obstrução biliar (ducto não ligado - DNL) usando o RT-PCR semiquantitativo e o qRT-PCR em tempo real. A metodologia do qRT-PCR em tempo real permitiu identificar mudanças no perfil de expressão gênica que não foram demonstrados pelo método semiquantitativo. O parênquima DL mostrou reação fibrogênica mais intensa, com aumento na expressão da alfa actina de músculo liso e TGF-beta1 após 7 dias. O parênquima DNL apresentou resposta fibrogênica tardia, com aumento da expressão da desmina 7 dias e 60 dias após a cirurgia, além de aumento da alfa-actina de músculo liso 60 dias após a cirurgia. O qRT-PCR em tempo real apresentou maior sensibilidade para identificar mudanças no perfil de expressão gênica em relação ao método convencional do RT-PCR semiquantitativo. Nossos resultados ajudam a esclarecer a dinâmica das alterações moleculares envolvidas na modulação da fibrogênese hepática no modelo experimental de ligadura seletiva do ducto biliar e pode ser diretamente aplicado para o estudo de estenoses biliares intra-hepáticas e atresia das vias biliares / The mechanisms responsible for liver fibrosis in childhood are poorly understood. Children suffering from biliary atresia, when submitted to the successful Kasai portoentostomy, become anicteric, but nonetheless develop cirrhosis in the long term. Similarly, the occurrence of intrahepatic biliary stenosis in the postoperative period of liver transplantation may lead to cirrhosis of the whole organ. Such facts suggest that endocrine or paracrine mechanisms are involved in hepatic fibrogenesis. To elucidate this process, the selective bile duct ligation model in young rats was developed in our laboratory. Using this model, we identified changes in the expression of smooth muscle alpha-actin both in the obstructed parenchyma and the hepatic parenchyma adjacent to the obstruction. However, the expression profiles of desmin, a protein present at high levels during activation of hepatic stellate cells and TGF-beta1, the main pro-fibrogenic cytokine, were unchanged when analyzed with semiquantitative RT-PCR. Thus, the molecular mechanisms involved in the modulation of hepatic fibrogenesis in this experimental model are not fully understood. The methodology of qRT-PCR (real time PCR) has previously been described as a more precise and sensitive method, allowing the detection of increased copy number of the gene while amplification occurs, whereas by semiquantitative RT-PCR analysis transcripts is only perfomed after the amplification step. This study aimed to evaluate the molecular changes in experimental model of selective bile duct ligation and compare the results between semiquantitative RT-PCR and real-time qRT-PCR methods. Selective biliary duct ligation was performed on Wistar rats with 21 days of life, the groups were separated according to the moment of death: 7 or 60 days after surgery. The expression of desmin, alpha-actin smooth muscle and TGF-beta1 was examined in tissue from hepatic parenchyma with biliary obstruction (duct ligation - DL) and in the adjacent hepatic parenchyma (duct non-ligated - DNL) using semiquantitative RT-PCR and real-time qRT-PCR. The methodology of the real-time qRT-PCR allowed to identify changes in gene expression profile that were not shown by semiquantitative method. The DL parenchyma showed a more severe fibrogenic reaction, with increased alpha-actin smooth muscle and TGF-beta1 expression after 7 days. The DNL parenchyma presented a later fibrotic response, with increased desmin expression 7 and 60 days after surgery, besides of increased alpha-actin smooth muscle 60 days after surgery. Real-time qRT-PCR was more sensitive to identify changes in gene expression profile comparated to the semiquantitative method. Our results help to clarify the dynamic of molecular changes involved in the modulation of hepatic fibrogenesis in an experimental model of selective bile duct ligation and can be directly applied to the study of intrahepatic biliary stenosis and biliary atresia

Os mecanismos responsáveis pela fibrose hepática na infância são pouco conhecidos. Crianças com atresia das vias biliares, quando submetidas a portoenterostomia a Kasai com sucesso, se tornam anictéricas mas mesmo assim desenvolvem cirrose a longo prazo. Da mesma forma, a ocorrência de estenoses biliares segmentares intra-hepáticas no pós-operatório de transplante hepático podem levar ao desenvolvimento de cirrose em todo o órgão. Tais fatos sugerem que mecanismos endócrinos ou parácrinos estejam envolvidos na fibrogênese hepática. Para elucidar este processo o modelo de ligadura seletiva das vias biliares em ratos jovens foi desenvolvido em nosso laboratório. Usando este modelo, identificamos mudanças na expressão do gene da alfa-actina de músculo liso, tanto no parênquima hepático obstruído como no parênquima hepático adjacente à obstrução. No entanto, o perfil de expressão gênica da desmina, uma proteína presente em níveis elevados durante a ativação das células estreladas hepáticas e o TGF-beta1, principal citocina pró-fibrogênica, não demostraram diferenças significantes quando analisados pelo método do RT-PCR semiquantitativo. Assim, os mecanismos moleculares envolvidos na modulação da fibrogênese hepática nesse modelo experimental não estão totalmente compreendidos. A metodologia do qRT-PCR (PCR em tempo real), têm sido previamente descrita como um método mais preciso e sensível, possibilitando a detecção do aumento do número de cópias do gene à medida que ocorre a amplificação, enquanto que o método do RT-PCR semiquantitativo a análise dos transcritos só é realizada após a etapa da amplificação. O objetivo deste estudo foi avaliar as alterações moleculares no modelo experimental de ligadura seletiva das vias biliares e comparar os resultados obtidos entre os métodos do RT-PCR semiquantitativo e do qRT-PCR em tempo real. Foi realizada a ligadura seletiva do ducto biliar em ratos Wistar com 21 dias de vida, os grupos foram separados de acordo com o momento da morte: 7 ou 60 dias após a cirurgia. A expressão da desmina, alfa actina de músculo liso e TGF-beta1 foi avaliada no tecido do parênquima hepático com obstrução biliar (ducto ligado - DL) e no parênquima hepático adjacente à obstrução biliar (ducto não ligado - DNL) usando o RT-PCR semiquantitativo e o qRT-PCR em tempo real. A metodologia do qRT-PCR em tempo real permitiu identificar mudanças no perfil de expressão gênica que não foram demonstrados pelo método semiquantitativo. O parênquima DL mostrou reação fibrogênica mais intensa, com aumento na expressão da alfa actina de músculo liso e TGF-beta1 após 7 dias. O parênquima DNL apresentou resposta fibrogênica tardia, com aumento da expressão da desmina 7 dias e 60 dias após a cirurgia, além de aumento da alfa-actina de músculo liso 60 dias após a cirurgia. O qRT-PCR em tempo real apresentou maior sensibilidade para identificar mudanças no perfil de expressão gênica em relação ao método convencional do RT-PCR semiquantitativo. Nossos resultados ajudam a esclarecer a dinâmica das alterações moleculares envolvidas na modulação da fibrogênese hepática no modelo experimental de ligadura seletiva do ducto biliar e pode ser diretamente aplicado para o estudo de estenoses biliares intra-hepáticas e atresia das vias biliares / The mechanisms responsible for liver fibrosis in childhood are poorly understood. Children suffering from biliary atresia, when submitted to the successful Kasai portoentostomy, become anicteric, but nonetheless develop cirrhosis in the long term. Similarly, the occurrence of intrahepatic biliary stenosis in the postoperative period of liver transplantation may lead to cirrhosis of the whole organ. Such facts suggest that endocrine or paracrine mechanisms are involved in hepatic fibrogenesis. To elucidate this process, the selective bile duct ligation model in young rats was developed in our laboratory. Using this model, we identified changes in the expression of smooth muscle alpha-actin both in the obstructed parenchyma and the hepatic parenchyma adjacent to the obstruction. However, the expression profiles of desmin, a protein present at high levels during activation of hepatic stellate cells and TGF-beta1, the main pro-fibrogenic cytokine, were unchanged when analyzed with semiquantitative RT-PCR. Thus, the molecular mechanisms involved in the modulation of hepatic fibrogenesis in this experimental model are not fully understood. The methodology of qRT-PCR (real time PCR) has previously been described as a more precise and sensitive method, allowing the detection of increased copy number of the gene while amplification occurs, whereas by semiquantitative RT-PCR analysis transcripts is only perfomed after the amplification step. This study aimed to evaluate the molecular changes in experimental model of selective bile duct ligation and compare the results between semiquantitative RT-PCR and real-time qRT-PCR methods. Selective biliary duct ligation was performed on Wistar rats with 21 days of life, the groups were separated according to the moment of death: 7 or 60 days after surgery. The expression of desmin, alpha-actin smooth muscle and TGF-beta1 was examined in tissue from hepatic parenchyma with biliary obstruction (duct ligation - DL) and in the adjacent hepatic parenchyma (duct non-ligated - DNL) using semiquantitative RT-PCR and real-time qRT-PCR. The methodology of the real-time qRT-PCR allowed to identify changes in gene expression profile that were not shown by semiquantitative method. The DL parenchyma showed a more severe fibrogenic reaction, with increased alpha-actin smooth muscle and TGF-beta1 expression after 7 days. The DNL parenchyma presented a later fibrotic response, with increased desmin expression 7 and 60 days after surgery, besides of increased alpha-actin smooth muscle 60 days after surgery. Real-time qRT-PCR was more sensitive to identify changes in gene expression profile comparated to the semiquantitative method. Our results help to clarify the dynamic of molecular changes involved in the modulation of hepatic fibrogenesis in an experimental model of selective bile duct ligation and can be directly applied to the study of intrahepatic biliary stenosis and biliary atresia