TY - JOUR
T1 - Proteomics Analysis of the Cardiac Myofilament Subproteome Reveals Dynamic Alterations in Phosphatase Subunit Distribution
AU - Yin, Xiaoke
AU - Cuello, Friederike
AU - Mayr, Ursula
AU - Hao, Zhiqi
AU - Hornshaw, Martin
AU - Ehler, Elisabeth
AU - Avkiran, Metin
AU - Mayr, Manuel
PY - 2010/3
Y1 - 2010/3
N2 - Myofilament proteins are responsible for cardiac contraction. The myofilament subproteome, however, has not been comprehensively analyzed thus far. In the present study, cardiomyocytes were isolated from rodent hearts and stimulated with endothelin-1 and isoproterenol, potent inducers of myofilament protein phosphorylation. Subsequently, cardiomyocytes were "skinned," and the myofilament subproteome was analyzed using a high mass accuracy ion trap tandem mass spectrometer (LTQ Orbitrap XL) equipped with electron transfer dissociation. As expected, a small number of myofilament proteins constituted the majority of the total protein mass with several known phosphorylation sites confirmed by electron transfer dissociation. More than 600 additional proteins were identified in the cardiac myofilament subproteome, including kinases and phosphatase subunits. The proteomic comparison of myofilaments from control and treated cardiomyocytes suggested that isoproterenol treatment altered the subcellular localization of protein phosphatase 2A regulatory subunit B56 alpha. Immunoblot analysis of myocyte fractions confirmed that beta-adrenergic stimulation by isoproterenol decreased the B56 alpha content of the myofilament fraction in the absence of significant changes for the myosin phosphatase target subunit isoforms 1 and 2 (MYPT1 and MYPT2). Furthermore, immunolabeling and confocal microscopy revealed the spatial redistribution of these proteins with a loss of B56 alpha from Z-disc and M-band regions but increased association of MYPT1/2 with A-band regions of the sarcomere following beta-adrenergic stimulation. In summary, we present the first comprehensive proteomics data set of skinned cardiomyocytes and demonstrate the potential of proteomics to unravel dynamic changes in protein composition that may contribute to the neurohormonal regulation of myofilament contraction. Molecular & Cellular Proteomics 9: 497-509, 2010.
AB - Myofilament proteins are responsible for cardiac contraction. The myofilament subproteome, however, has not been comprehensively analyzed thus far. In the present study, cardiomyocytes were isolated from rodent hearts and stimulated with endothelin-1 and isoproterenol, potent inducers of myofilament protein phosphorylation. Subsequently, cardiomyocytes were "skinned," and the myofilament subproteome was analyzed using a high mass accuracy ion trap tandem mass spectrometer (LTQ Orbitrap XL) equipped with electron transfer dissociation. As expected, a small number of myofilament proteins constituted the majority of the total protein mass with several known phosphorylation sites confirmed by electron transfer dissociation. More than 600 additional proteins were identified in the cardiac myofilament subproteome, including kinases and phosphatase subunits. The proteomic comparison of myofilaments from control and treated cardiomyocytes suggested that isoproterenol treatment altered the subcellular localization of protein phosphatase 2A regulatory subunit B56 alpha. Immunoblot analysis of myocyte fractions confirmed that beta-adrenergic stimulation by isoproterenol decreased the B56 alpha content of the myofilament fraction in the absence of significant changes for the myosin phosphatase target subunit isoforms 1 and 2 (MYPT1 and MYPT2). Furthermore, immunolabeling and confocal microscopy revealed the spatial redistribution of these proteins with a loss of B56 alpha from Z-disc and M-band regions but increased association of MYPT1/2 with A-band regions of the sarcomere following beta-adrenergic stimulation. In summary, we present the first comprehensive proteomics data set of skinned cardiomyocytes and demonstrate the potential of proteomics to unravel dynamic changes in protein composition that may contribute to the neurohormonal regulation of myofilament contraction. Molecular & Cellular Proteomics 9: 497-509, 2010.
U2 - 10.1074/mcp.M900275-MCP200
DO - 10.1074/mcp.M900275-MCP200
M3 - Article
VL - 9
SP - 497
EP - 509
JO - MOLECULAR AND CELLULAR PROTEOMICS
JF - MOLECULAR AND CELLULAR PROTEOMICS
IS - 3
ER -