QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 8 April 2003
doi: 10.1242/jcs.00447

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jenniskens, G. J. Articles by van Kuppevelt, T. H. Search for Related Content PubMed PubMed Citation Articles by Jenniskens, G. J. Articles by van Kuppevelt, T. H.

Disturbed Ca²⁺ kinetics in N-deacetylase/N-sulfotransferase-1 defective myotubes

Guido J. Jenniskens^1,*, Maria Ringvall², Werner J. H. Koopman³, Johan Ledin², Lena Kjellén², Peter H. G. M. Willems³, Erik Forsberg², Jacques H. Veerkamp¹ and Toin H. van Kuppevelt^1,

¹ Department of Biochemistry 194, University Medical Center, NCMLS, 6500 HB Nijmegen, The Netherlands
² Department of Medical Biochemistry and Microbiology, Uppsala University, S-751 23 Uppsala, Sweden
³ Department of Biochemistry 160/Microscopical Imaging Center, University Medical Center, NCMLS, 6500 HB Nijmegen, The Netherlands
^* Present address: Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

View larger version (75K): [in a new window]   Fig. 1. Reduction of HS epitopes in NDST-1–/– skeletal muscle. Immunofluorescence staining for (a,c,e,g) HS-epitope AO4B05 and (b,d,f,h) acetylcholine receptor clusters in intercostal muscle of E18.5 embryos of (a,b,e,f) wildtype, (c,d) NDST-1–/–and (g,h) NDST-2–/–. Overall staining in NDST-1–/– is significantly reduced, compared to wild type and NDST-2–/– staining, except for the BL in the NMJs and their direct vicinity (n=5; see also Fig. 2a,b). Bar, 50 µm.

View larger version (144K): [in a new window]   Fig. 2. The distribution of the dihydropyridine receptor (L-type Ca2+ channel) and the perlecan core protein are unaffected in NDST-1–/– skeletal muscle. Immunofluorescence staining for (a,b) HS-epitope AO4B05, (c,d) DHPR, and (e,f) perlecan core protein in (a,c,e) wild type and (b,d,f) NDST-1–/– skeletal muscle. Overall staining for HS epitope AO4B05 is significantly reduced in (b) NDST-1–/–, compared to (a) wild-type muscle, whereas staining (c,d) for DHPR and (e,f) for perlecan remains virtually unaltered. Note the persistence of staining in neural tissue in (b) (n=3). Bar, 75 µm.

View larger version (11K): [in a new window]   Fig. 3. Alteration of N- and O-sulfation levels in skeletal muscle of NDST-1–/– mouse. The percentage of N-acetylated HS disaccharides (NAc) is strongly increased in skeletal muscle of NDST-1–/– mice (open bars), compared to wild type (solid bars). The N-acetylated HS disaccharides fraction may contain a minor portion of chondroitin sulfate. Furthermore, the percentages of N-sulfated (NS), N-sulfated/2-O-sulfated (NS2S), and N-sulfated/6-O-sulfated/2-O-sulfated (NS6S2S) disaccharides are decreased in NDST-1–/– mouse, whereas the percentages of 6-O-sulfated (6S), N-sulfated/6-O-sulfated (NS6S), and 2-O-sulfated/6-O-sulfated (2S6S) disaccharides are largely unaltered. (Wildtype, n=2; NDST-1–/–, n=3).

View larger version (66K): [in a new window]   Fig. 4. Reduction of HS epitopes in NDST-1–/– primary muscle cell culture. Immunofluorescence staining for (a,c,e,g) HS-epitope AO4B05 and (b,d,f,h) acetylcholine receptor clusters in (a,b,e,f) wildtype, (c,d) NDST-1–/– and (g,h) NDST-2–/– primary muscle culture after three days of differentiation. HS staining is absent in NDST-1–/–derived myotubes and mononuclear cells. In contrast, NDST-2–/– cultures are not affected. Myotubes in all cultures show spontaneous acetylcholine receptors clusters (arrows; n=5). Bar, 50 µm.

View larger version (17K): [in a new window]   Fig. 5. Disturbed Ca2+ kinetics in NDST-1-defective myotubes. Primary myoblast cultures of wild-type and NDST-1–/– embryos were differentiated for three days, and Indo-1 ratios of electrically induced Ca2+ spikes were recorded using a high-speed UV confocal laser scanning microscope. Normalized Indo-1 ratios of individual wild-type and NDST-1–/– primary myotubes were averaged and plotted against time, thus generating representative ratio traces for both genotypes. Each point represents the average value (wild type, n=12; NDST-1–/–, n=8).