title: Co-expression of SERCA isoforms, phospholamban and sarcolipin in human skeletal muscle fibers.
abstract: Sarcolipin (SLN) and phospholamban (PLN) inhibit the activity of sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs) by reducing their apparent affinity for Ca(2+). A ternary complex between SLN, PLN, and SERCAs results in super-inhibition of SERCA activity. Analysis of skeletal muscle homogenate has limited our current understanding of whether SLN and PLN regulate SERCA1a, SERCA2a, or both in skeletal muscle and whether SLN and PLN are co-expressed in skeletal muscle fibers. Biopsies from human vastus lateralis were analyzed through single fiber Western blotting and immunohisto/fluorescence staining to circumvent this limitation. With a newly generated SLN antibody, we report for the first time that SLN protein is present in human skeletal muscle. Addition of the SLN antibody (50 µg) to vastus lateralis homogenates increased the apparent Ca(2+) affinity of SERCA (K Ca, pCa units) (-Ab, 5.85 ± 0.02 vs. +Ab, 5.95 ± 0.02) and maximal SERCA activity (μmol/g protein/min) (-Ab, 122 ± 6.4 vs. +Ab, 159 ± 11) demonstrating a functional interaction between SLN and SERCAs in human vastus lateralis. Specifically, our results suggest that although SLN and PLN may preferentially regulate SERCA1a, and SERCA2a, respectively, physiologically they both may regulate either SERCA isoform. Furthermore, we show that SLN and PLN co-immunoprecipitate in human vastus lateralis homogenate and are simultaneously expressed in 81% of the fibers analyzed with Western blotting which implies that super-inhibition of SERCA may exist in human skeletal muscle. Finally, we demonstrate unequivocally that mouse soleus contains PLN protein suggesting that super-inhibition of SERCA may also be important physiologically in rodent skeletal muscle.
authors.affiliation: Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: Val A
authors.initials: VA
authors.lastname: Fajardo
authors.affiliation: Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: Eric
authors.initials: E
authors.lastname: Bombardier
authors.affiliation: Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: Chris
authors.initials: C
authors.lastname: Vigna
authors.affiliation: Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: Tahira
authors.initials: T
authors.lastname: Devji
authors.affiliation: Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: Darin
authors.initials: D
authors.lastname: Bloemberg
authors.affiliation: Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: Daniel
authors.initials: D
authors.lastname: Gamu
authors.affiliation: Department of Physiology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Anthony O
authors.initials: AO
authors.lastname: Gramolini
authors.affiliation: Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: Joe
authors.initials: J
authors.lastname: Quadrilatero
authors.affiliation: Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: A Russell
authors.initials: AR
authors.lastname: Tupling
mesh_terms: D000293:Adolescent; D000328:Adult; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes; D008297:Male; D051379:Mice; D018656:Muscle Fibers, Fast-Twitch / Q000378:metabolism; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D018657:Muscle Fibers, Slow-Twitch / Q000378:metabolism; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000378:metabolism; D011485:Protein Binding; D011510:Proteolipids / Q000235:genetics / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D055815:Young Adult; D000099211:Phospholamban
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D007527:Isoenzymes; D009124:Muscle Proteins; D018995:Myosin Heavy Chains; D011510:Proteolipids; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban; C077258:sarcolipin