ALTERATIONS OF ECCENTRICALLY EVOKED PROTEIN SYNTHESIS IN ISOLATED RAT SOLEUS FOLLOWING GRAVITATIONAL UNLOADING: ROLE OF FAK
-
1
Institute of Biomedical Problems (RAS), Russia
Introduction
Skeletal muscle mass is critical for muscle function and can vary depending on the load, activity or pathological conditions. Prolonged periods of muscle inactivity as a result of gravitational unloading can lead to muscle disuse atrophy, a complex process, which is characterized by a decrease in muscle fiber cross-sectional area, protein content, muscle strength and an increase in muscle fatigue (Thomason and Booth 1990, Zhang et al. 2007). Precise mechanisms by which mechanical load transduce to anabolic and catabolic response (mechanotransduction) remain undefined, despite of fundamental dependence of its development and maintenance from mechanical stimuli (Hornberger 2011). mTORC1 is a key regulator of mRNA translation and, consequently, protein synthesis. Accordingly, mTORC1 is able to influence both the translational efficiency (the rate of mRNA translation) and the translational capacity (rate of ribosome biogenesis) (Mahoney et al. 2009). Noteworthy that, mTORC1 activation by a different types of mechanical stimuli was shown in a skeletal muscle using various models (cell-culture, whole muscle in vivo and ex vivo models) (Goodman 2014). There are also different molecules that can be proposed as skeletal muscle mechanosensor – stretch-activated channels (SAC) (McBride 2003), focal adhesion-integrin system (Fluck et al. 1999), sarcomeric proteins (Gautel 2011) and others. Focal adhesion kinase can be interconnected with a phosphoinositide 3-kinase (PI3K) – FAK tyr398 phosphorylation leads to its binding with SH2 domain of 85kDa PI3K subunit, and this, in turn, to PI3K activation, and ,therefore, to the whole PI3K, AKT, p70S6k pathway activation (Chen et al. 1996). In the present study we analyzed an impact of FAK inhibition on realization of mechanical signal in unloaded skeletal muscle after eccentric contraction.
Materials and methods
Unloading of the hindlimbs was induced by using a standard rodent hindlimb suspension/unloading model (Morey-Holton and Globus 2002). Wistar rats weighing 220±5 g were randomly divided into the following groups: Control (C), Hindlimb Suspension for 7 days (HS) and HS plus FAK inhibitor – PD562,271 (HS+PD) (Roberts et al. 2008). HS was followed by ex vivo bout of Eccentric Exercise (ECC). Isolated rat soleus muscle was placed in Krebs–Henseleit solution with constant perfusion of carbogen (95% O2 + 5% CO2) and maintaining of the temperature at 37°C. One end of the muscle was connected to a dynamometer/stress generator by silk threads through tendons and another to a fixed retainer. The muscle was then stretched to the optimum length (L0). Eccentric contractions were performed using electric field stimulation (80 V, 50 Hz for 3 s). The muscle was stretched to 15% of L0 during stimulation (the time of stretching and returning to the previous length was 100 ms) (Burry et al. 2007). Upon completion of the EC, muscles were subjected to Western blot analyses in order to determine the content of phosphorylated forms of the key anabolic markers. Phosphorylation status as a proxy of activation of the signaling proteins was expressed relative to the total amount of each protein. The rate of the protein synthesis was analyzed using SUnSET (surface sensing of translation) technique (Goodman et al. 2011). Intact muscle was compared to a muscle after ECC. The significance of the differences between groups was determined by analysis of the variance (ANOVA method).
Results and discussion
Western blot analysis revealed a significant decrease in phosphorylated/total ratios of p70s6k (marker of mTORC1 activity) and its substrate S6rp after 7-day HS. SUnSET measurements also showed a significant 40% decrease in the protein synthesis rate following 7 days of HS (p<0.05). We didn’t observe any significant differences in the HS group compared to the HS+PD group. An increase in protein synthesis after ECC was significantly higher in the C group compared to the HS group (+127%), and didn’t differ from that of the HS+PD group (Fig.1). We also observed similar effect for p70s6k and S6rp (Fig.2) phosphorylated/total ratios. Earlier we showed that inhibition of SAC in the unloaded rat soleus can lead to attenuated anabolic response to eccentric contractions (Mirzoev et al. 2018). Nevertheless, SAC inhibition did not lead to a complete AKT/mTOR pathway inactivation. This fact suggests that there are other mechanosensors which can detect and transduce mechanical signals. One of such mechanosensors is believed to be FAK. In the present study, FAK inhibition in the unloaded rat soleus muscle prevented the attenuated anabolic response following ECC. We speculate that this effect could be associated with the disturbance of possible reciprocal relationships between the two mechanosensors (SAC and FAK).
Conclusion
In summary, the results of the study indicate that inhibition of FAK activity in the unloaded rat soleus muscle may prevent a diminished anabolic response to mechanical stimuli.
Acknowledgements
The study was supported by the Basic Research Program of the SSC RF - IBMP RAS (topic no. 65.3) and RFBR grant # 16-34-60055
References
Burry M, Hawkins D, Spangenburg EE. 2007. Lengthening contractions differentially affect p70s6k phosphorylation compared to isometric contractions in rat skeletal muscle. European journal of applied physiology. Jul;100:409-415.
Chen HC, Appeddu PA, Isoda H, Guan JL. 1996. Phosphorylation of tyrosine 397 in focal adhesion kinase is required for binding phosphatidylinositol 3-kinase. The Journal of biological chemistry. Oct 18;271:26329-26334.
Fluck M, Carson JA, Gordon SE, Ziemiecki A, Booth FW. 1999. Focal adhesion proteins FAK and paxillin increase in hypertrophied skeletal muscle. The American journal of physiology. Jul;277:152-162.
Gautel M. 2011. Cytoskeletal protein kinases: titin and its relations in mechanosensing. Pflugers Archiv : European journal of physiology. Jul;462:119-134.
Goodman CA. 2014. The role of mTORC1 in regulating protein synthesis and skeletal muscle mass in response to various mechanical stimuli. Reviews of physiology, biochemistry and pharmacology.166:43-95.
Goodman CA, Mabrey DM, Frey JW, Miu MH, Schmidt EK, Pierre P, Hornberger TA. 2011. Novel insights into the regulation of skeletal muscle protein synthesis as revealed by a new nonradioactive in vivo technique. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. Mar;25:1028-1039.
Hornberger TA. 2011. Mechanotransduction and the regulation of mTORC1 signaling in skeletal muscle. The international journal of biochemistry & cell biology. Sep;43:1267-1276.
Mahoney SJ, Dempsey JM, Blenis J. 2009. Cell signaling in protein synthesis ribosome biogenesis and translation initiation and elongation. Progress in molecular biology and translational science.90:53-107.
McBride TA. 2003. Stretch-activated ion channels and c-fos expression remain active after repeated eccentric bouts. Journal of applied physiology. Jun;94:2296-2302.
Mirzoev TM, Tyganov SA, Shenkman BS. 2018. The role of stretch-activated channels in mTORC1 signaling in rat m. soleus in response to a mechanical stimulus following hindlimb unloading. Rossiiskii fiziologicheskii zhurnal imeni IM Sechenova.104:217-225.
Morey-Holton ER, Globus RK. 2002. Hindlimb unloading rodent model: technical aspects. Journal of applied physiology. Apr;92:1367-1377.
Roberts WG, Ung E, Whalen P, Cooper B, Hulford C, Autry C, Richter D, Emerson E, Lin J, Kath J, et al. 2008. Antitumor activity and pharmacology of a selective focal adhesion kinase inhibitor, PF-562,271. Cancer research. Mar 15;68:1935-1944.
Thomason DB, Booth FW. 1990. Atrophy of the soleus muscle by hindlimb unweighting. Journal of applied physiology. Jan;68:1-12.
Zhang P, Chen X, Fan M. 2007. Signaling mechanisms involved in disuse muscle atrophy. Medical hypotheses.69:310-321.
Keywords:
soleus muscle,,
FAK,
Mechanotransduction,
Eccentric contraction (ECC),
hindlimb suspension (unloading),
mTORC1
Conference:
39th ISGP Meeting & ESA Life Sciences Meeting, Noordwijk, Netherlands, 18 Jun - 22 Jun, 2018.
Presentation Type:
Extended abstract
Topic:
Bones and Muscles
Citation:
Tyganov
S,
Mirzoev
T,
Rozhkov
S and
Shenkman
B
(2019). ALTERATIONS OF ECCENTRICALLY EVOKED PROTEIN SYNTHESIS IN ISOLATED RAT SOLEUS FOLLOWING GRAVITATIONAL UNLOADING: ROLE OF FAK.
Front. Physiol.
Conference Abstract:
39th ISGP Meeting & ESA Life Sciences Meeting.
doi: 10.3389/conf.fphys.2018.26.00005
Copyright:
The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers.
They are made available through the Frontiers publishing platform as a service to conference organizers and presenters.
The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated.
Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed.
For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions.
Received:
02 Dec 2018;
Published Online:
16 Jan 2019.
*
Correspondence:
Mr. Sergey Tyganov, Institute of Biomedical Problems (RAS), Moscow, Russia, sentackle@yandex.ru