title: Soluble miniagrin enhances contractile function of engineered skeletal muscle.
abstract: Neural agrin plays a pleiotropic role in skeletal muscle innervation and maturation, but its specific effects on the contractile function of aneural engineered muscle remain unknown. In this study, neonatal rat skeletal myoblasts cultured within 3-dimensional engineered muscle tissue constructs were treated with 10 nM soluble recombinant miniagrin and assessed using histological, biochemical, and functional assays. Depending on the treatment duration and onset time relative to the stage of myogenic differentiation, miniagrin was found to induce up to 1.7-fold increase in twitch and tetanus force amplitude. This effect was associated with the 2.3-fold up-regulation of dystrophin gene expression at 6 d after agrin removal and enhanced ACh receptor (AChR) cluster formation, but no change in cell number, expression of muscle myosin, or important aspects of intracellular Ca(2+) handling. In muscle constructs with endogenous ACh levels suppressed by the application of α-NETA, miniagrin increased AChR clustering and twitch force amplitude but failed to improve intracellular Ca(2+) handling and increase tetanus-to-twitch ratio. Overall, our studies suggest that besides its synaptogenic function that could promote integration of engineered muscle constructs in vivo, neural agrin can directly promote the contractile function of aneural engineered muscle via mechanisms distinct from those involving endogenous ACh.
authors.affiliation: Department of Anesthesia and Medicine and Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Weining
authors.initials: W
authors.lastname: Bian
authors.forename: Nenad
authors.initials: N
authors.lastname: Bursac
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D018171:Agrin / Q000494:pharmacology* / Q000502:physiology; D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects; D002452:Cell Count; D002478:Cells, Cultured; D016189:Dystrophin / Q000235:genetics; D007537:Isometric Contraction / Q000187:drug effects / Q000502:physiology; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D032448:Myoblasts, Skeletal / Q000187:drug effects / Q000502:physiology; D009218:Myosins / Q000378:metabolism; D051381:Rats; D011950:Receptors, Cholinergic / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D012995:Solubility; D023822:Tissue Engineering; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D018171:Agrin; D016189:Dystrophin; D011950:Receptors, Cholinergic; D011994:Recombinant Proteins; D009218:Myosins; D000109:Acetylcholine
references.citation: Biomaterials. 2011 May;32(14):3575-83
references.pmid: 21324402
references.citation: Cell Tissue Res. 2003 Nov;314(2):263-74
references.pmid: 12955494
references.citation: Biophys J. 2008 Sep 15;95(6):3009-16
references.pmid: 18502803
references.citation: Heart Rhythm. 2007 May;4(5):619-26
references.pmid: 17467631
references.citation: Mol Ther. 2011 Jan;19(1):165-71
references.pmid: 20924363
references.citation: Proc Natl Acad Sci U S A. 2002 Oct 1;99(20):12979-84
references.pmid: 12271128
references.citation: J Biol Chem. 1996 Mar 1;271(9):5231-6
references.pmid: 8617807
references.citation: Cell. 1996 May 17;85(4):525-35
references.pmid: 8653788
references.citation: J Neurosci. 2003 Jan 15;23(2):539-49
references.pmid: 12533614
references.citation: J Biol Chem. 1998 Jan 9;273(2):736-43
references.pmid: 9422725
references.citation: Nat Med. 2007 May;13(5):642-8
references.pmid: 17417652
references.citation: Curr Protoc Stem Cell Biol. 2009 Jun;Chapter 1:Unit 1F.8
references.pmid: 19536758
references.citation: In Vitro Cell Dev Biol Anim. 2006 Mar-Apr;42(3-4):75-82
references.pmid: 16759152
references.citation: Development. 1993 Mar;117(3):1153-61
references.pmid: 8391977
references.citation: Cell. 1991 Nov 29;67(5):909-16
references.pmid: 1659950
references.citation: J Muscle Res Cell Motil. 2010 Jul;31(1):59-69
references.pmid: 20490629
references.citation: J Cell Biol. 1987 Dec;105(6 Pt 1):2471-8
references.pmid: 2826489
references.citation: Proc Natl Acad Sci U S A. 2008 Aug 12;105(32):11406-11
references.pmid: 18685098
references.citation: Cell. 1996 May 17;85(4):513-23
references.pmid: 8653787
references.citation: J Cell Biol. 1997 May 5;137(3):671-83
references.pmid: 9151673
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: J Cell Biol. 2001 Jun 25;153(7):1441-52
references.pmid: 11425874
references.citation: Am J Physiol Cell Physiol. 2008 Jan;294(1):C66-73
references.pmid: 18003748
references.citation: Biol Cell. 1997 Oct;89(7):413-34
references.pmid: 9561721
references.citation: Nat Rev Neurosci. 2001 Nov;2(11):791-805
references.pmid: 11715056
references.citation: J Cell Biol. 1988 May;106(5):1703-12
references.pmid: 3372592
references.citation: Proc Natl Acad Sci U S A. 1980 Aug;77(8):4717-20
references.pmid: 6933518
references.citation: Proc Natl Acad Sci U S A. 2005 Aug 2;102(31):11088-93
references.pmid: 16043708
references.citation: Mol Cell Neurosci. 1997;9(4):254-63
references.pmid: 9268504
references.citation: News Physiol Sci. 2003 Dec;18:222-5
references.pmid: 14614153
references.citation: Exerc Sport Sci Rev. 2003 Apr;31(2):73-8
references.pmid: 12715970
references.citation: Biomaterials. 2009 Mar;30(7):1401-12
references.pmid: 19070360
references.citation: Neuron. 2001 Jul 19;31(1):15-22
references.pmid: 11498047
references.citation: Neuromuscul Disord. 1998 Aug;8(6):362-70
references.pmid: 9713852
references.citation: Eur J Appl Physiol. 2006 Sep;98(1):1-21
references.pmid: 16896733
references.citation: Clin Plast Surg. 1999 Oct;26(4):647-56, ix-x
references.pmid: 10553219
references.citation: Nat Protoc. 2008;3(6):1101-8
references.pmid: 18546601
references.citation: Biophys J. 2009 May 6;96(9):3873-85
references.pmid: 19413993
references.citation: Cell Mol Neurobiol. 2009 Feb;29(1):123-31
references.pmid: 18807173
references.citation: Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):5985-90
references.pmid: 8650206
references.citation: Brain Res Dev Brain Res. 2000 Feb 7;119(2):289-95
references.pmid: 10675780
references.citation: Cold Spring Harb Symp Quant Biol. 1990;55:407-18
references.pmid: 1966767
references.citation: Proc Natl Acad Sci U S A. 2000 Apr 25;97(9):4950-5
references.pmid: 10781103
references.citation: J Physiol. 1995 Dec 15;489 ( Pt 3):791-803
references.pmid: 8788943
references.citation: Nat Protoc. 2009;4(10):1522-34
references.pmid: 19798085
references.citation: Cell Tissue Res. 2000 Jun;300(3):447-57
references.pmid: 10928275
references.citation: Development. 2002 Dec;129(23):5449-60
references.pmid: 12403715
references.citation: J Pharmacol Exp Ther. 1988 Apr;245(1):72-80
references.pmid: 3361452
references.citation: J Cell Biol. 2001 Feb 5;152(3):435-50
references.pmid: 11157973
references.citation: J Physiol. 2005 Oct 1;568(Pt 1):171-80
references.pmid: 16037088