Phenotypic knock out of heparan sulfates in myotubes impairs excitation-induced calcium spiking.
Little is known about the physiological functions of heparan sulfates (HSs), which are present in the basal lamina surrounding skeletal muscle fibers. Here, we present a new system in which HS is phenotypically knocked out by endogenous expression of epitope-specific anti-HS antibodies. Single-chain antibodies, containing an immunoglobulin leader peptide, were produced by using various expression systems. Antibodies were detected in the Golgi apparatus, the site of HS biosynthesis. Likewise, the HS-degrading enzyme heparanase was expressed. Endogenous expression of antibodies or heparanase in myoblasts resulted in HS-defective myotubes. Excitability and calcium kinetics of HS-defective myotubes were severely compromised, as determined by analysis of electrically induced calcium spikes via video-speed UV confocal laser scanning microscopy. Phenotypically knocking out of individual HS epitopes resulted in specific effects on excitability and calcium kinetics. These data indicate important roles for HSs in skeletal muscle calcium kinetics.
Jenniskens
Guido J
GJ
Department of Biochemistry 194, University Medical Center, NCMLS, 6500 HB Nijmegen, The Netherlands.
Koopman
Werner J H
WJ
Willems
Peter H G M
PH
Pecker
Iris
I
Veerkamp
Jacques H
JH
van Kuppevelt
Toin H
TH
D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D005786:Gene Expression Regulation; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006497:Heparan Sulfate / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D014162:Transfection
D000906:Antibodies; D002118:Calcium; D049452:Green Fluorescent Proteins; D006497:Heparan Sulfate; D008164:Luminescent Proteins