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 Ca2+-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 ± 51.0versus 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 Ca2+transport activity at pCa 5 by 31% with no significant change in apparent Ca2+ affinity (6.36 ± 0.07versus 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 Ca2+ stores. Sarcolipin (SLN) is an inhibitor of sarco(endo)plasmic reticulum Ca2+-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 ± 51.0versus 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 Ca2+transport activity at pCa 5 by 31% with no significant change in apparent Ca2+ affinity (6.36 ± 0.07versus 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 Ca2+ stores. Sarcolipin (SLN) 1The abbreviations used are: SLN, sarcolipin; SERCA, sarco (endo)plasmic reticulum Ca2+-ATPase; PLN, phospholamban; NF-SLN, sarcolipin tagged N-terminally with a FLAG epitope; MOPS, 4-morpholinepropanesulfonic acid.1The abbreviations used are: SLN, sarcolipin; SERCA, sarco (endo)plasmic reticulum Ca2+-ATPase; PLN, phospholamban; NF-SLN, sarcolipin tagged N-terminally with a FLAG epitope; MOPS, 4-morpholinepropanesulfonic acid. is a 31-amino acid transmembrane protein which co-purifies with the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1a) (1MacLennan D.H. Yip C.C. Iles G.H. Seeman P. Cold Spring Harbor Symp. Quant. Biol. 1972; 37: 469-478Google Scholar, 2Wawrzynow A. Theibert J.L. Murphy C. Jona I. Martonosi A. Collins J.H. Arch. Biochem. Biophys. 1992; 298: 620-623Google Scholar). Like SERCA1a, SLN is expressed highly in both human and rabbit fast twitch muscle and to a lower extent in slow twitch and cardiac muscle, where phospholamban (PLN) and SERCA2a are highly expressed (3Fujii J. Lytton J. Tada M. MacLennan D.H. FEBS Lett. 1988; 227: 51-55Google Scholar), and in trace amounts in pancreas and prostate (4Odermatt A. Taschner P.E. Scherer S.W. Beatty B. Khanna V.K. Cornblath D.R. Chaudhry V. Yee W.C. Schrank B. Karpati G. Breuning M.H. Knoers N. MacLennan D.H. Genomics. 1997; 45: 541-553Google Scholar). In rat, however, the pattern of expression is different (5Gayan-Ramirez G. Vanzeir L. Wuytack F. Decramer M. J. Physiol. 2000; 524: 387-397Google Scholar), because SLN is expressed to a low extent in fast twitch extensor digitorum longus muscle and nearly 18-fold higher in both diaphragm (mixed fast and slow) and cardiac muscles. Our initial studies indicated that SLN decreased the apparent affinity of SERCA1a for Ca2+ and increased maximal transport activity at high Ca2+ concentrations (6Odermatt A. Becker S. Khanna V.K. Kurzydlowski K. Leisner E. Pette D. MacLennan D.H. J. Biol. Chem. 1998; 273: 12360-12369Google Scholar). Our more recent studies have confirmed that co-expression of NF-SLN with SERCAs in HEK-293 cells decreases the apparent affinity of both SERCA1a and SERCA2a for Ca2+ but did not confirm that NF-SLN increases maximal transport activity of SERCA1a in high Ca2+ (7Asahi M. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 2002; 277: 26725-26728Google Scholar). The apparent increase in maximal transport activity of SERCA1a observed earlier (6Odermatt A. Becker S. Khanna V.K. Kurzydlowski K. Leisner E. Pette D. MacLennan D.H. J. Biol. Chem. 1998; 273: 12360-12369Google Scholar) could be explained by an underestimate in the enzyme-linked immunosorbant assay used in that study to measure the amount of SERCA1a (7Asahi M. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 2002; 277: 26725-26728Google Scholar). NF-SLN was found to be a more effective inhibitor of SERCA2a than of SERCA1a, being almost equal to PLN in its ability to decrease apparent Ca2+ affinity and, in contrast to PLN, even decreasing maximal transport activity at pCa 5 (7Asahi M. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 2002; 277: 26725-26728Google Scholar). PLN is a well characterized regulator of SERCA2a activity in cardiac muscle (8Simmerman H.K. Jones L.R. Physiol. Rev. 1998; 78: 921-947Google Scholar). Inhibitory interactions between PLN and SERCA2a result in a decrease in the apparent affinity of SERCA2a for Ca2+ (9Kimura Y. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1996; 271: 21726-21731Google Scholar) with no effect on the maximal rate of Ca2+ uptake by SERCA2a (7Asahi M. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 2002; 277: 26725-26728Google Scholar, 10Odermatt A. Taschner P.E. Khanna V.K. Busch H.F. Karpati G. Jablecki C.K. Breuning M.H. MacLennan D.H. Nat. Genet. 1996; 14: 191-194Google Scholar, 11Morris G.L. Cheng H.C. Colyer J. Wang J.H. J. Biol. Chem. 1991; 266: 11270-11275Google Scholar). Inhibitory interactions can be reversed by elevation of cytosolic Ca2+ or by phosphorylation of Ser16 and Thr17 in PLN cytosolic domain 1A (12Simmerman H.K. Collins J.H. Theibert J.L. Wegener A.D. Jones L.R. J. Biol. Chem. 1986; 261: 13333-13341Google Scholar). Several studies have shown that PLN is a major regulator of left ventricular basal contractile parameters and their responses to β-agonists (13Sham J.S. Jones L.R. Morad M. Am. J. Physiol. 1991; 261: H1344-H1349Google Scholar, 14Luo W. Grupp I.L. Harrer J. Ponniah S. Grupp G. Duffy J.J. Doetschman T. Kranias E.G. Circ. Res. 1994; 75: 401-409Google Scholar, 15Koss K.L. Kranias E.G. Circ. Res. 1996; 79: 1059-1063Google Scholar, 16Kadambi V.J. Ponniah S. Harrer J.M. Hoit B.D. Dorn G.W. II Walsh R.A. Kranias E.G. J. Clin. Invest. 1996; 97: 533-539Google Scholar). Ablation of PLN is associated with significant increases in cardiac contractility and left ventricular systolic function (14Luo W. Grupp I.L. Harrer J. Ponniah S. Grupp G. Duffy J.J. Doetschman T. Kranias E.G. Circ. Res. 1994; 75: 401-409Google Scholar, 15Koss K.L. Kranias E.G. Circ. Res. 1996; 79: 1059-1063Google Scholar), whereas the 2-fold overexpression of wild-type PLN in transgenic mice had an inhibitory effect on both the kinetics of Ca2+ transients and contractile parameters in ventricular myocytes and impaired basal left ventricular systolic function in vivo (16Kadambi V.J. Ponniah S. Harrer J.M. Hoit B.D. Dorn G.W. II Walsh R.A. Kranias E.G. J. Clin. Invest. 1996; 97: 533-539Google Scholar). Structural similarities betweenSLN and PLN genes and SLN and PLN protein sequences indicate that the two genes are members of a family (4Odermatt A. Taschner P.E. Scherer S.W. Beatty B. Khanna V.K. Cornblath D.R. Chaudhry V. Yee W.C. Schrank B. Karpati G. Breuning M.H. Knoers N. MacLennan D.H. Genomics. 1997; 45: 541-553Google Scholar). The physiological function of SLN has not been evaluated, either in skeletal muscle or in cardiac muscle. Because NF-SLN and PLN affect SERCA2a function in a similar fashion in vitro, it is likely that SLN and PLN would affect muscle contractility in a similar fashion. In this study, we expressed NF-SLN in rat soleus muscle by intramuscular injection and electrotransfer of rabbit NF-SLN cDNA to explore the possibility that SLN can regulate SERCA2a activity and slow twitch soleus muscle contractility just as PLN can regulate cardiac contractility. We found that NF-SLN reduces peak isometric force, slows the rates of contraction and relaxation, and increases susceptibility to fatigue. Ca2+ uptake in postnuclear homogenates was also We that NF-SLN can muscle contractile function by inhibiting SERCA function and basal Ca2+ in the sarcoplasmic for were and and the expression was and a for of co-immunoprecipitation and were FLAG and the were the W. E. Biochem. Biophys. Res. 2000; Scholar) was a the SERCA2a and was The was in E. L. MacLennan D.H. Clin. Biol. Res. Scholar). was on soleus and P. N. Biochem. Scholar), the being as a for SLN is expressed highly in rat (5Gayan-Ramirez G. Vanzeir L. Wuytack F. Decramer M. J. Physiol. 2000; 524: 387-397Google Scholar). and were by at and of was in of a of at in the of inhibitor and SLN The was in a of 50 with with of the and and and 5 of was for at was at for and for was at for by a at for The were on The of their with SERCA1a, and NF-SLN, and the of cells have been in earlier (6Odermatt A. Becker S. Khanna V.K. Kurzydlowski K. Leisner E. Pette D. MacLennan D.H. J. Biol. Chem. 1998; 273: 12360-12369Google Scholar, Y. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1997; Scholar, M. Y. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. Scholar). The of the rabbit NF-SLN cDNA was (6Odermatt A. Becker S. Khanna V.K. Kurzydlowski K. Leisner E. Pette D. MacLennan D.H. J. Biol. Chem. 1998; 273: 12360-12369Google Scholar). NF-SLN is a protein of SLN with a FLAG at its that not its function (6Odermatt A. Becker S. Khanna V.K. Kurzydlowski K. Leisner E. Pette D. MacLennan D.H. J. Biol. Chem. 1998; 273: 12360-12369Google Scholar). The NF-SLN was into and of the expression and of the NF-SLN cDNA into rat soleus muscle was the of J. D. J.M. P. D. B. D. S. A. Scholar). ± were with a of and soleus muscle was and with a of of in a the soleus muscle the hindlimb was with NF-SLN, and the soleus muscle the contralateral hindlimb was with an equal of the expression 2 were by two to a in the soleus muscle were and the were to a muscle force was assessed in the soleus muscle hindlimb a of stimulation to The were a of and and of as to the and the stimulation have been F. J. J. Physiol. 1997; Scholar). and both twitch and tetanic force were muscle with the of and with a at V. stimulation was a and force were a to a and the and and was for force 5 o) for peak twitch force (P t) was tetanic force (P o) at a stimulation of 50 and and are peak force and peak rates of contraction (+dF/dt) and relaxation the of NF-SLN on soleus susceptibility to muscle was for 3 a of at 50 once every 2 are expressed as of were by the of the of muscle contractile soleus were and in 5 and and with a at maximal with two The homogenates were in for to the homogenates were on and at in an for 5 to and The postnuclear was assessed for protein the with as a and was used for Western blotting was to the expression of NF-SLN, PLN, and SERCA1a G. W. C. M. Grupp I.L. J.S. Kranias E.G. Circ. Res. 1996; 79: Scholar) in rat soleus that were either with NF-SLN or the expression of of the postnuclear were to and or and by and to with the were with both to for protein and either or for at and with with were with and with an was for a were in on with a HEK-293 cells that had been with either NF-SLN or PLN (7Asahi M. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 2002; 277: 26725-26728Google Scholar) as and for NF-SLN and PLN, Western blotting was on homogenates soleus that expressed NF-SLN and HEK-293 cells that expressed both NF-SLN and either SERCA1a or SERCA2a. and HEK-293 were to the by to and for NF-SLN and either SERCA1a or SERCA2a the was to the of SERCA1a and SERCA2a to NF-SLN in both rat soleus and HEK-293 confirm between the SERCA expressed in rat soleus E. L. M. L. Wuytack F. Biochem. J. 1996; Scholar), and NF-SLN, co-immunoprecipitation of SERCA2a and NF-SLN was as M. Y. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. Scholar) of postnuclear homogenates at and a protein FLAG The of SERCA2a associated with NF-SLN in the was by Western blotting and the Ca2+ transport activity in postnuclear homogenates at was in of a 5 5 5 and of protein as Y. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1997; Scholar, M. Y. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. Scholar). The were by the were an for a for The for maximal transport activity that at pCa 5 were the and for protein The maximal transport activity are as of which was to a the were in The are as the ± was by between and soleus with was of rat soleus muscle and rat were used in to SLN is expressed in We did not to SLN expression in either rat soleus or cardiac but we were to confirm expression in both of not NF-SLN cDNA was into the expression by the in the confirm the of NF-SLN in rat soleus with NF-SLN we Western blotting on postnuclear homogenates with the NF-SLN cDNA and with NF-SLN was in that were with NF-SLN cDNA NF-SLN expression was it was to with the to the confirm that NF-SLN was with SERCA2a in the co-immunoprecipitation of SERCA2a with NF-SLN was the that physical interactions between SERCA2a and NF-SLN in soleus with NF-SLN The expression of both SERCA2a and SERCA1a were compared between soleus with NF-SLN cDNA and were no in either SERCA2a or SERCA1a expression between NF-SLN and 3 We confirmed an earlier E. A. Biophys. 2000; Scholar) in which and were used to show that PLN is not expressed in rat soleus muscle 3 was of to a measure of the amount of NF-SLN that in soleus muscle injection of NF-SLN was not to because NF-SLN was not to however, to the of NF-SLN in to the amount of SERCA in soleus muscle and to the results with the between NF-SLN and SERCA expressed in HEK-293 cells where physiological are Western blotting was on homogenates soleus that expressed NF-SLN and on HEK-293 cells that expressed NF-SLN with either SERCA1a or SERCA2a. and HEK-293 were to the by to and for NF-SLN and either SERCA1a or SERCA2a the was to the of SERCA1a and SERCA2a to NF-SLN in the rat soleus was also used to of SERCA1a to NF-SLN and SERCA2a to NF-SLN in HEK-293 cells that expressed NF-SLN and either SERCA1a or SERCA2a. of soleus that expressed NF-SLN that expressed the amount of NF-SLN to SERCA1a and SERCA2a and we the in HEK-293 cells at we could that the of SERCA2a to NF-SLN expression in soleus muscle was and the of SERCA1a to NF-SLN was we that SERCA1a and SERCA2a for of the SERCA in soleus muscle Lytton J. Am. J. Physiol. Scholar), the of SERCA1a SERCA2a to NF-SLN would be the of endogenous SERCA expression in soleus muscle, we are of the amount of NF-SLN that has on SERCA function in HEK-293 SLN is expressed in rat soleus muscle, at an in it is the effect of NF-SLN overexpression on of an of SLN expression that we are in physiological studies of soleus muscle. We the of muscle contractile function and SERCA function for the in which NF-SLN was highly the physiological of NF-SLN expression in slow twitch skeletal muscle, isometric contractile both twitch and soleus with NF-SLN cDNA were in and compared with contractile assessed in NF-SLN reduced peak twitch force (P t) by compared with The maximal twitch rate of contraction (+dF/dt) was reduced by and the maximal twitch rate of relaxation (−dF/dt) was reduced by with NF-SLN expression tetanic force (P which at a stimulation of 50 was reduced by 5 tetanic was reduced by and tetanic was reduced by with NF-SLN expression 5 of NF-SLN on soleus isometric tetanic injection and electrotransfer of NF-SLN into rat isometric tetanic properties were in as and compared with tetanic properties assessed in contralateral which were with the expression contractile properties that expressed the of NF-SLN and are a tetanic force for a soleus muscle that was with NF-SLN and a one peak tetanic (P o) at 50 Hz in and soleus that expressed NF-SLN maximal tetanic rate of contraction (+dF/dt) and rate of relaxation (−dF/dt) in and soleus that expressed NF-SLN versus susceptibility to was assessed by stimulation of the muscle the The force and of contraction that are of were in soleus NF-SLN compared with expressed as a of was reduced by ± and was reduced by ± in compared with in of ± and in of ± in In the peak relaxation rate was with to a similar ± in NF-SLN muscle and ± in muscle. homogenates were to a of and for Ca2+ of Ca2+ uptake to the of NF-SLN expression on SERCA NF-SLN reduced Ca2+ uptake a of Ca2+ pCa to pCa 5 NF-SLN reduced at pCa to pCa 5 that maximal transport activity was reduced by compared with was no significant however, in apparent Ca2+ expressed in pCa (6.36 ± 0.07versus 6.39 ± ± These in studies of co-expression of NF-SLN with SERCA1a or SERCA2a in HEK-293 where a significant change was observed in apparent Ca2+ affinity (7Asahi M. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 2002; 277: 26725-26728Google Scholar). is likely to in the of NF-SLN expression compared with the expression of In studies with HEK-293 cells (7Asahi M. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 2002; 277: 26725-26728Google Scholar), we changes in of to pCa the of NF-SLN to SERCA1a or SERCA2a used in were the were reduced to no in were observed in the and absence of NF-SLN is that the of SERCA to SLN even overexpression of the physiological function of SLN in we expressed NF-SLN in rat soleus one hindlimb the contralateral as a Our results confirm recent in studies that SLN as an inhibitor of SERCA function a to pCa in a decreased apparent Ca2+ affinity and lower maximal transport activity (7Asahi M. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 2002; 277: 26725-26728Google Scholar). decrease in Ca2+ uptake is in muscle contractile which is impaired expression of NF-SLN in slow twitch skeletal muscle. In both the kinetics and of contraction were reduced with NF-SLN and soleus were was more in NF-SLN compared with NF-SLN expression in slow twitch skeletal muscle is similar to a of PLN overexpression in the at in of its on muscle contractility. We were to NF-SLN in rat soleus a of injection and electrotransfer of plasmid with cDNA injection was to NF-SLN because we were to NF-SLN in rat soleus that were with NF-SLN not is in with studies that in vivo to plasmid into skeletal J. D. J.M. P. D. B. D. S. A. Scholar, M. P. N. P. D. K. Scholar, K. F. N. Y. I. M. F. T. M. J. Scholar, S. L. J. E. Scholar). Our co-immunoprecipitation and Ca2+ transport in postnuclear homogenates indicate that expressed NF-SLN both and with SERCA2a in rat soleus sarcoplasmic reticulum We confirmed that rat soleus not PLN, which is that co-expression of PLN and NF-SLN with either SERCA2a or SERCA1a is for SERCA function (7Asahi M. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 2002; 277: 26725-26728Google Scholar). We have also shown that rat soleus is a in which to the function of SLN with SERCA2a and The of this study an and because muscle function and just 3 at the where peak expression of a introduced cDNA is observed J. D. J.M. P. D. B. D. S. A. Scholar), changes in the expression of Ca2+ would be In we did not significant changes in the expression of either SERCA2a or SERCA1a with NF-SLN in this study was that we were to SLN and NF-SLN expression in muscle. an SLN has not been to we could not SLN protein or expression between and The of highly NF-SLN also it to of NF-SLN enzyme-linked immunosorbant SLN is in rat soleus muscle, that SLN is also expressed in this muscle. we could show that NF-SLN expression to SERCA expression in soleus was of the that was for of SERCA in HEK-293 the and between and NF-SLN soleus were in of contractile function and Ca2+ of the effect of overexpression of is that endogenous SLN protein are for physiological function and that increases in SLN expression can result in impaired The of this study was that NF-SLN expression can as an inhibitor of SERCA function in vivo and, soleus contractile the between NF-SLN and PLN because PLN overexpression can SERCA function to cardiac contractility (16Kadambi V.J. Ponniah S. Harrer J.M. Hoit B.D. Dorn G.W. II Walsh R.A. Kranias E.G. J. Clin. Invest. 1996; 97: 533-539Google Scholar) and, to a skeletal muscle contractility Grupp I.L. N. Kranias E.G. J. Biol. Chem. 1997; Scholar). The of NF-SLN on force rate of and rate of relaxation observed in this study are similar to the of wild-type PLN overexpression in transgenic mice (16Kadambi V.J. Ponniah S. Harrer J.M. Hoit B.D. Dorn G.W. II Walsh R.A. Kranias E.G. J. Clin. Invest. 1996; 97: 533-539Google Scholar) and rat ventricular myocytes K. H.K. P. A. Physiol. Genomics. Scholar) and to the overexpression of the and of PLN in E. F. Y. S. Hoit B.D. Kranias E.G. MacLennan D.H. J. Biol. Chem. 2000; Scholar). In of contraction and rates of and in myocytes were The of PLN overexpression on cardiac contractile function were to changes in and kinetics that were observed (16Kadambi V.J. Ponniah S. Harrer J.M. Hoit B.D. Dorn G.W. II Walsh R.A. Kranias E.G. J. Clin. Invest. 1996; 97: 533-539Google Scholar, K. H.K. P. A. Physiol. Genomics. Scholar). These changes in Ca2+ and muscle contractile parameters are to result reduced sarcoplasmic reticulum Ca2+ uptake and of SERCA2a function and of sarcoplasmic reticulum Ca2+ (16Kadambi V.J. Ponniah S. Harrer J.M. Hoit B.D. Dorn G.W. II Walsh R.A. Kranias E.G. J. Clin. Invest. 1996; 97: 533-539Google Scholar, K. H.K. P. A. Physiol. Genomics. Scholar, E. F. Y. S. Hoit B.D. Kranias E.G. MacLennan D.H. J. Biol. Chem. 2000; Scholar). the amount of Ca2+ in the sarcoplasmic reticulum rat myocytes with 2-fold overexpression of PLN was reduced by K. H.K. P. A. Physiol. Genomics. Scholar). for the that a similar is for the changes in contractile function that expression of NF-SLN in rat soleus muscle, the maximal Ca2+ transport activity was reduced by 31% in postnuclear homogenates muscle NF-SLN compared with The this study was by NF-SLN, but the Ca2+ uptake rate was lower with NF-SLN expression compared with a of low to high lower rate of Ca2+ the in the of NF-SLN would the of the Ca2+ in the sarcoplasmic and a reduced Ca2+ could in skeletal muscle B. J. Biol. Chem. 2002; 277: Scholar), for the effect of NF-SLN on isometric twitch and tetanic contractile properties observed in this Because SLN is expressed in the an this study and an earlier study (7Asahi M. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 2002; 277: 26725-26728Google Scholar) is that overexpression of SLN in the would have the to cardiac contractile In a study, the overexpression of PLN in fast twitch skeletal muscle impaired the relaxation rate with no effect on the rate of contraction or peak twitch Grupp I.L. N. Kranias E.G. J. Biol. Chem. 1997; Scholar), in contrast to the of overexpression of PLN in the (16Kadambi V.J. Ponniah S. Harrer J.M. Hoit B.D. Dorn G.W. II Walsh R.A. Kranias E.G. J. Clin. Invest. 1996; 97: 533-539Google Scholar). is because fast twitch skeletal muscle also SLN, and we have shown that co-expression of NF-SLN and PLN is for SERCA1a function (7Asahi M. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 2002; 277: 26725-26728Google Scholar), the expressed in fast twitch skeletal muscle Lytton J. Am. J. Physiol. Scholar). In study it is not to the of SERCA that were by the of NF-SLN endogenous SLN, was it to the of SERCA that were by the of expressed PLN expressed SLN in the study of Grupp I.L. N. Kranias E.G. J. Biol. Chem. 1997; Scholar). SERCA are more in fast twitch muscle compared with slow twitch skeletal muscle L. Rev. Physiol. Biochem. Scholar). a in both studies is that the were not to of SERCA to We also assessed the of NF-SLN expression on soleus susceptibility to an that slow twitch fast twitch with tetanic force was lower in soleus that expressed indicated that was a of force and reduced rate of contraction in soleus the change in relaxation rate was similar between soleus that expressed NF-SLN and that is no interaction between NF-SLN expression and in to the of relaxation that with J. Physiol. Scholar). These results that NF-SLN expression was for the observed in susceptibility to fatigue. In the between force and is a which changes in pCa to changes in force J. Physiol. Scholar). is that soleus NF-SLN, as to were in that where in Ca2+ would to in force, that basal sarcoplasmic reticulum Ca2+ and Ca2+ are to be lower in soleus that is an of skeletal muscle function characterized by of muscle relaxation N. J. Scholar). We have associated in the with of but not with (4Odermatt A. Taschner P.E. Scherer S.W. Beatty B. Khanna V.K. Cornblath D.R. Chaudhry V. Yee W.C. Schrank B. Karpati G. Breuning M.H. Knoers N. MacLennan D.H. Genomics. 1997; 45: 541-553Google Scholar, A. Kurzydlowski K. MacLennan D.H. J. Biol. Chem. 1996; 271: Scholar). for in the SLN in that were not has not in or sequences in the SLN (4Odermatt A. Taschner P.E. Scherer S.W. Beatty B. Khanna V.K. Cornblath D.R. Chaudhry V. Yee W.C. Schrank B. Karpati G. Breuning M.H. Knoers N. MacLennan D.H. Genomics. 1997; 45: 541-553Google Scholar). the of the results of this study, however, SLN expression not be in SLN as a for In we have found that expression of NF-SLN in rat soleus results in a significant in muscle contractility and increased susceptibility to fatigue. Ca2+ uptake in postnuclear homogenates was also that NF-SLN as an inhibitor of SERCA function in These results that overexpression of SLN has the to skeletal muscle We are to for the of the and to for to and of the rat contractile and

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