Cardiac-specific overexpression of sarcolipin in phospholamban null mice impairs myocyte function that is restored by phosphorylation.
Sarcolipin (SLN) inhibits the cardiac sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2a) by direct binding and is superinhibitory if it binds as a binary complex with phospholamban (PLN). To demonstrate whether overexpression of SLN in the heart might impair cardiac function directly, transgenic (TG) mice with cardiac-specific overexpression of NF-SLN (SLN tagged at its N terminus with the FLAG epitope) were generated on a phospholamban (PLN) null (PLN KO) background. In NF-SLN TG/PLN KO cardiac microsomes, the apparent affinity of SERCA2a for Ca2+ was decreased compared with non-TG littermate PLN KO hearts. Analyses of isolated NF-SLN/PLN KO cardiomyocytes revealed impaired cardiac contractility, reduced calcium transient peak amplitude, and slower decay kinetics compared to PLN KO animals. In these cardiomyocytes, isoproterenol restored calcium dynamics to the levels seen in PLN KO. Invasive hemodynamic and echocardiographic analyses of NF-SLN/PLN KO mouse cardiac muscle in vivo showed no direct effects of NF-SLN overexpression when compared to PLN KO mice. A possible mechanism for the lack of effects in the whole heart may be a responsiveness to phosphorylation because we determined that NF-SLN can be phosphorylated in cardiomyocytes in response to isoproterenol, and we provide evidence that serine/threonine kinase 16 is a kinase that can phosphorylate NF-SLN. Site-directed mutagenesis showed that SLN Thr-5 is the target site for this kinase. These data show that overexpression of NF-SLN can inhibit SERCA2a in the absence of PLN and that the inhibition of SERCA2a is correlated with impairment of contractility and calcium cycling in cardiomyocytes.
Gramolini
Anthony O
AO
Banting and Best Department of Medical Research, Charles H. Best Institute, Heart and Stroke Richard Lewar Centre, and Department of Physiology, University of Toronto, Toronto, ON, Canada M5G 1L6. anthony.gramolini@utoronto.ca
Trivieri
Maria G
MG
Oudit
Gavin Y
GY
Kislinger
Thomas
T
Li
Wenping
W
Patel
Mikin M
MM
Emili
Andrew
A
Kranias
Evangelia G
EG
Backx
Peter H
PH
Maclennan
David H
DH
D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002316:Cardiotonic Agents / Q000494:pharmacology; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009200:Myocardial Contraction / Q000235:genetics / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000032:analysis / Q000378:metabolism; D011510:Proteolipids / Q000235:genetics / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D015533:Transcriptional Activation; D016276:Ventricular Function; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D002316:Cardiotonic Agents; D007545:Isoproterenol; D009124:Muscle Proteins; D010770:Phosphotransferases; D011510:Proteolipids; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban; C077258:sarcolipin