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Cigarette smoke exposure up-regulates Ubiquitin specific protease 19 in murine skeletal muscles as an adaptive response to prolonged ER stress
Department of Clinical Medicine, Örebro University, Örebro, Sweden.
Department of Clinical Medicine, Örebro University, Örebro, Sweden; School of Biomedical Sciences, Charles Sturt University, WaggaWagga, Australia.
Department of Clinical Medicine, Örebro University, Örebro, Sweden; Medical University of Giessen, Molecular Biology and Medicine of the Lung program, Giessen, Germany.
Örebro universitet, Institutionen för naturvetenskap och teknik.
Vise andre og tillknytning
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
Abstract [en]

Enhanced protein degradation via ubiquitin proteolytic system (UPS) was demonstrated to play an important role in the pathogenesis of cachexia syndrome and muscle wasting in patients with COPD and animal models of the disease. The role of cigarette smoke (CS) exposure in eliciting these abnormalities remains largely unknown. Usp19 is a member of UPS suggested to be involved in progressive muscle wasting in different catabolic conditions. However, factors regulating Usp19 expression, activity and correlation/s with CS-induced muscle atrophy remainunclear.

Methods: To address these questions, 129 SvJ mice were exposed to cigarette smoke for 6 months and the gastrocnemius muscles were collected. Expression levels of Usp19 as well as pivotal mediators of ER stress response have been studied using PCR, qPCR, western blot and immunofluorescence. Factors regulating muscle Usp19 expression were studied using in-silico analysis of Usp19 promoter as well as by stimulating C2C12 myocytes with different inducers of ER stress including hypoxia, TNF and tunicamycin. Finally, Usp19 expression was depleted in C2C12 myocytes using specific Usp19 siRNA quadriplex and the expression of pivotal myogenic regulators were analyzed.

Results: Usp19 mRNA expression was enhanced in skeletal muscles of CS-exposed mice. Concurrently, ER stress-associated Caspase 12 and Caspase 3 were activated in the CS-exposed group. Analysis of Usp19 promoter sequence revealed binding sites for ER stress response transcription factors such as HSF, STRE1 and AML1-α. Exposure of C2C12 myocytes to tunicamycin but not hypoxia elevated expression levels of Usp19. TNFstimulation elevated Usp19 protein expression but inhibited its RNA transcription in a dose- and time-dependent manner. Finally, Usp19 overexpression in tunicamycin-treated myocytes was accompanied by reduced expression of myosin heavy chain and tropomyosin and their levels were increased after knocking down Usp19 in C2C12 myocytes.

Conclusions: In summary, our data demonstrated elevated expression of Usp19 in skeletal muscles of CS-exposed 129 SvJ mice. Moreover, Usp19 overexpression was associated with muscle adaptations to ER stress and suppression of myogenesis. Taken together; our results might provide further insight into molecular mechanisms underlying development and progression of skeletal muscle abnormalities in response to chronic cigarette smoke exposure.

HSV kategori
Forskningsprogram
Biomedicin
Identifikatorer
URN: urn:nbn:se:oru:diva-38194OAI: oai:DiVA.org:oru-38194DiVA, id: diva2:758613
Tilgjengelig fra: 2014-10-27 Laget: 2014-10-27 Sist oppdatert: 2017-10-17bibliografisk kontrollert
Inngår i avhandling
1. Molecular mechanisms mediating development of pulmonary cachexia in COPD
Åpne denne publikasjonen i ny fane eller vindu >>Molecular mechanisms mediating development of pulmonary cachexia in COPD
2014 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Cigarette smoking (CS) represents the main causative agent underlying development and progress of COPD. Recently, involvement of CS in the pathogenesis of COPDassociated muscle abnormalities is becoming increasingly evident. Nevertheless, involved triggers and underlying mechanisms remain largely unknown. This study was conceived in order to examine effects of cigarette smoke exposure on skeletal muscle morphology, vascular supply and function. For this purpose, we have specifically designed murine COPD/emphysema model and gastrocnemius muscle was examined, while in vitro experiments were conducted using murine C2C12 skeletal muscle myocytes.

In addition to the mild emphysematous changes present in the lungs of CS-exposed mice, our results demonstrated evident signs of muscle atrophy reflected by decreased fiber cross-sectional area, profound fiber size variation and reduced body mass. Furthermore, we have observed impairment in terminal myogenesis and lower number of myonuclei in skeletal muscles of CS-exposed animals despite evident activation of muscle repair process. Additionally, our results demonstrate capillary rarefaction in skeletal muscles of CS-exposed animals which was associated with deregulation of hypoxia-angiogenesis signaling, reduced levels of angiogenic factors such as HIF1-α and VEGF and enhanced expression of VHL and its partner proteins PHD2 and Ube2D1. The results of our in-vitro experiments demonstrated that VHL and its ubiquitination machinery can be synergistically regulated by TNF and hypoxia consequentially impairing angiogenic potential of skeletal muscle myocytes. Finally, we have shown that CS elicits chronic ER stress in murine skeletal muscles which is associated with activation of ERAD and apoptotic pathways as mirrored by elevated expression of Usp19, caspase 12 and caspase 3 in skeletal muscles of CSexposed animals. Moreover, molecular and morphological alterations in CS-exposed mice resulted in impairment of muscle function as reflected by their impaired exercise capacity.

Taken together, from our results it is evident that cigarette smoke exposure elicits set of morphological, vascular and functional changes highly resembling those observed in COPD. Additionally, CS induces wide range of molecular alterations and signaling pathway deregulations suggesting profound effects of cigarette smoke exposure on skeletal muscle cell homeostasis.

sted, utgiver, år, opplag, sider
Örebro: Örebro university, 2014. s. 76
Serie
Örebro Studies in Medicine, ISSN 1652-4063 ; 107
Emneord
COPD, cachexia, atrophy, cigarette smoke, myogenesis, angiogenesis
HSV kategori
Forskningsprogram
Biomedicin; Biokemi
Identifikatorer
urn:nbn:se:oru:diva-36104 (URN)978-91-7529-031-7 (ISBN)
Disputas
2014-09-16, Universitetssjukhuset, hörsal C2, Södra Grev Rosengatan, Örebro, 09:15 (engelsk)
Opponent
Tilgjengelig fra: 2014-08-25 Laget: 2014-08-25 Sist oppdatert: 2018-01-11bibliografisk kontrollert

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