Overexpression of sarcolipin decreases myocyte contractility and calcium transient.
OBJECTIVE
Sarcolipin (SLN) is a novel 31-amino-acid protein associated with the sarcoplasmic reticulum (SR) whose function in cardiac muscle is poorly defined. In this study, we tested the hypothesis that SLN is a regulator of SR Ca(2+) transport function by overexpressing SLN in adult rat ventricular myocytes which express low levels of SLN.


METHODS
Expression of SLN mRNA in rat tissues was analyzed by Northern blot as well by RT-PCR analysis. To define the role of SLN in cardiac muscle contractility, we overexpressed SLN in adult rat ventricular myocytes using adenoviral gene transfer techniques. Localization of SLN in the adult rat ventricular myocytes was determined using confocal microscopy. Myocyte contractility and calcium transients were measured using edge detection and Fura 2AM.


RESULTS
Our results demonstrate that overexpression of SLN decreased the cell shortening significantly when compared to control myocytes, whereas the time to peak contraction was not altered. In addition, SLN overexpression prolonged the time of 50% relaxation. Calcium transient analysis shows that time to 50% decay of [Ca(2+) ]i was markedly prolonged in SLN-overexpressing myocytes (control -245.0+/-3.78 vs. SLN -199.0+/-3.25 ms, p<0.001). However, there were no significant differences in peak amplitudes of [Ca(2+)](i) between SLN-overexpressing and control myocytes. We further demonstrate that SLN is localized within the SR membrane similar to PLB and SR Ca(2+) ATPase. Co-immunoprecipitation studies indicate that SLN can physically interact with phospholamban.


CONCLUSIONS
We conclude that SLN may play an important role in regulating the SR calcium ATPase pump, possibly by interacting with phospholamban.
Babu
Gopal J
GJ
Department of Physiology and Cell Biology, The Ohio State University College of Medicine and Public Health, Columbus, OH 43210, USA.
Zheng
Zhaolun
Z
Natarajan
Poornima
P
Wheeler
Debra
D
Janssen
Paul M
PM
Periasamy
Muthu
M
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015152:Blotting, Northern / Q000379:methods; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D048429:Cell Size; D015870:Gene Expression; D005822:Genetic Vectors / Q000008:administration & dosage; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism*; D011510:Proteolipids / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D014161:Transduction, Genetic / Q000379:methods; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D009124:Muscle Proteins; D011510:Proteolipids; D000099211:Phospholamban; C077258:sarcolipin