Sarcolipin overexpression in rat slow twitch muscle inhibits sarcoplasmic reticulum Ca2+ uptake and impairs contractile function.
Sarcolipin (SLN) is an inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs) in vitro, but its function in vivo has not been defined. NF-SLN cDNA (SLN tagged N-terminally with a FLAG epitope) was introduced into rat soleus muscle in one hindlimb by plasmid injection and electrotransfer. Western blotting showed expression and co-immunoprecipitation showed physical interaction between NF-SLN and SERCA2a. Contractile properties and SERCA2a function were assessed and compared with vector-injected contralateral soleus muscles. NF-SLN reduced both peak twitch force (P(t)) (123.9 +/- 12.5 versus 69.8 +/- 8.9 millinewtons) and tetanic force (P(o)) (562.3 +/- 51.0 versus 300.7 +/- 56.9 millinewtons) and reduced both twitch and tetanic rates of contraction (+dF/dt) and relaxation (-dF/dt) significantly. Repetitive stimulation (750-ms trains at 50 Hz once every 2 s for 3 min) showed that NF-SLN increased susceptibility to fatigue. These changes in contractile function were observed in the absence of endogenous phospholamban, and NF-SLN had no effect on either SERCA2a or SERCA1a expression levels. NF-SLN also decreased maximal Ca(2+) transport activity at pCa 5 by 31% with no significant change in apparent Ca(2+) affinity (6.36 +/- 0.07 versus 6.39 +/- 0.08 pCa units). These results show that NF-SLN expression impairs muscle contractile function by inhibiting SERCA function and diminishing sarcoplasmic reticulum Ca(2+) stores.
Tupling
A Russell
AR
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
Asahi
Michio
M
MacLennan
David H
DH
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004558:Electric Stimulation; D006801:Humans; D008297:Male; D009119:Muscle Contraction / Q000502:physiology*; D018763:Muscle Fatigue; D018657:Muscle Fibers, Slow-Twitch / Q000502:physiology*; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011510:Proteolipids / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D009124:Muscle Proteins; D011510:Proteolipids; D011993:Recombinant Fusion Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban; C077258:sarcolipin