title: Absence of γ-sarcoglycan alters the response of p70S6 kinase to mechanical perturbation in murine skeletal muscle.
abstract: BACKGROUND
  The dystrophin glycoprotein complex (DGC) is located at the sarcolemma of muscle fibers, providing structural integrity. Mutations in and loss of DGC proteins cause a spectrum of muscular dystrophies. When only the sarcoglycan subcomplex is absent, muscles display severe myofiber degeneration, but little susceptibility to contractile damage, suggesting that disease occurs not by structural deficits but through aberrant signaling, namely, loss of normal mechanotransduction signaling through the sarcoglycan complex. We extended our previous studies on mechanosensitive, γ-sarcoglycan-dependent ERK1/2 phosphorylation, to determine whether additional pathways are altered with the loss of γ-sarcoglycan.
  
  
  METHODS
  We examined mechanotransduction in the presence and absence of γ-sarcoglycan, using C2C12 myotubes, and primary cultures and isolated muscles from C57Bl/6 (C57) and γ-sarcoglycan-null (γ-SG(-/-)) mice. All were subjected to cyclic passive stretch. Signaling protein phosphorylation was determined by immunoblotting of lysates from stretched and non-stretched samples. Calcium dependence was assessed by maintaining muscles in calcium-free or tetracaine-supplemented Ringer's solution. Dependence on mTOR was determined by stretching isolated muscles in the presence or absence of rapamycin.
  
  
  RESULTS
  C2C12 myotube stretch caused a robust increase in P-p70S6K, but decreased P-FAK and P-ERK2. Neither Akt nor ERK1 were responsive to passive stretch. Similar but non-significant trends were observed in C57 primary cultures in response to stretch, and γ-SG(-/-) cultures displayed no p70S6K response. In contrast, in isolated muscles, p70S6K was mechanically responsive. Basal p70S6K activation was elevated in muscles of γ-SG(-/-) mice, in a calcium-independent manner. p70S6K activation increased with stretch in both C57 and γ-SG(-/-) isolated muscles, and was sustained in γ-SG(-/-) muscles, unlike the transient response in C57 muscles. Rapamycin treatment blocked all of p70S6K activation in stretched C57 muscles, and reduced downstream S6RP phosphorylation. However, even though rapamycin treatment decreased p70S6K activation in stretched γ-SG(-/-) muscles, S6RP phosphorylation remained elevated.
  
  
  CONCLUSIONS
  p70S6K is an important component of γ-sarcoglycan-dependent mechanotransduction in skeletal muscle. Our results suggest that loss of γ-sarcoglycan uncouples the response of p70S6K to stretch and implies that γ-sarcoglycan is important for inactivation of this pathway. Overall, we assert that altered load-sensing mechanisms exist in muscular dystrophies where the sarcoglycans are absent.
authors.affiliation: Department of Anatomy and Cell Biology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA ; Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Catherine
authors.initials: C
authors.lastname: Moorwood
authors.affiliation: Department of Anatomy and Cell Biology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA ; Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, PA, USA ; Current address: Department of Physiology, Medical School, National and Kapodistrian University of Athens, Goudi, Athens, Greece.
authors.forename: Anastassios
authors.initials: A
authors.lastname: Philippou
authors.affiliation: Department of Anatomy and Cell Biology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA ; Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Janelle
authors.initials: J
authors.lastname: Spinazzola
authors.affiliation: Department of Anatomy and Cell Biology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Keyser
authors.affiliation: Department of Anatomy and Cell Biology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA ; Current address: Department of Dermatology and Cutaneous Biology, Jefferson University College of Medicine, Philadelphia, PA, USA.
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Macarak
authors.affiliation: Department of Anatomy and Cell Biology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA ; Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Elisabeth R
authors.initials: ER
authors.lastname: Barton
keywords: ERK1/2; Limb girdle muscular dystrophy; Load-sensing; Mechano-sensing; Mechanotransduction; S6K; Sarcoglycan; Sarcoglycanopathies; p70S6 kinase; p70S6K