Proteomics Analysis of the Cardiac Myofilament Subproteome Reveals Dynamic Alterations in Phosphatase Subunit Distribution

Abstract

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 B56α. Immunoblot analysis of myocyte fractions confirmed that β-adrenergic stimulation by isoproterenol decreased the B56α 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 B56α from Z-disc and M-band regions but increased association of MYPT1/2 with A-band regions of the sarcomere following β-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.

Macrophage MicroRNA-155 Promotes Cardiac Hypertrophy and Failure

“…24 Media were subjected to mass spectroscopic proteomics analyses as described 25 or used for rat ventricular cardiomyocytes culturing after dialysis in low-serum rat ventricular cardiomyocyte media using Slide-A-Lyzer Dialysis Cassettes (1/100, 3.5 kDa; Thermo Fisher Scientific, Waltham, MA).…”

Cardiac Function Is Regulated by B56α-mediated Targeting of Protein Phosphatase 2A (PP2A) to Contractile Relevant Substrates

“…At a depolarizing potential of ؊5 mV, the I Ca,L current density was decreased by 28% after administration of ISO in TG cardiomyocytes. In addition, the ISO-stimulated phosphorylation of phospholamban at Ser 16 was reduced by 27% in TG hearts. Thus, the increased PP2A-B56␣ activity in TG hearts is localized to specific subcellular sites leading to the dephosphorylation of important contractile proteins.…”

“…co-localized with the myofilaments), but was excluded from the nucleus (15). Analysis of myocardial fractions revealed that B56␣ was decreased in the myofilament fraction and increased in the cytosol after ␤-adrenergic stimulation by isoproterenol (ISO) (16). These dynamic changes in PP2A-B56␣ targeting may contribute to neurohumoral regulation of cardiac performance under physiological conditions and in the diseased heart, as demonstrated for sepsis-associated cardiac dysfunction (17).…”

Protein Phosphorylation and Signal Transduction in Cardiac Thin Filaments

“…The major phosphatases controlling thin filament protein phosphorylation are PP1 and PP2A. Both of these phosphatases have been reported to have a Z-disc localization (73,74), placing them in close proximity to the A-and I-band regions of the sarcomere and indicating that their localization may be strain-sensitive. In fact, recent studies (74) indicate that that the B56a unit of the PP2A complex localizes to the Z-disc but moves away with ␤-adrenergic stimulation, whereas light chain 2 phosphatase does not.…”

“…Both of these phosphatases have been reported to have a Z-disc localization (73,74), placing them in close proximity to the A-and I-band regions of the sarcomere and indicating that their localization may be strain-sensitive. In fact, recent studies (74) indicate that that the B56a unit of the PP2A complex localizes to the Z-disc but moves away with ␤-adrenergic stimulation, whereas light chain 2 phosphatase does not. Our studies (26) showing a decrease in tropomyosin phosphorylation in hearts with constitutively active p38␣ demonstrated co-localization of this MAPK with ␣-actinin at the Z-disc, as well as protein phosphatases (PP2␣ and PP2␤).…”