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Results: 5

1.
Figure 4

Figure 4. cMyBP-C phosphorylation and cardiac function. From: A Critical Function for Ser-282 in Cardiac Myosin Binding Protein-C Phosphorylation and Cardiac Function.

A, B, Phosphorylation status of Ser-273, Ser-282 and Ser-302 sites before and after dobutamine treatment was determined by Western blots using phospho-specific antibodies (n=5). C, Contractile function was measured at baseline and dobutamine (32ng/ml min). Maximal rate of ventricular contraction (dP/dtmax) and relaxation (dP/dtmin). Data are presented as mean±S.E (n=6). *P<0.05, **P<0.005 compared with NTG; ¶P<0.05, ¶¶P<0.005, ¶¶¶P<0.0005 compared with (t/t), §P<0.05 compared with basal (absence of dobutamine stimulation) measurements.

Sakthivel Sadayappan, et al. Circ Res. ;109(2):141-150.
2.
Figure 3

Figure 3. Phenotypic analyses of sarcomere architecture. From: A Critical Function for Ser-282 in Cardiac Myosin Binding Protein-C Phosphorylation and Cardiac Function.

A, Incorporation of Myc-tagged mutant cMyBP-C was confirmed by immunofluorescent staining of cMyBP-C with either an anti-myc (A) or anti-cMyBP-C antibody (B). C, Longitudinal sections of the heart stained with hematoxylin/eosin (20x) or D, Masson-trichrome (20x) to assess fibrosis. Note the areas of disrupted myocyte organization and fibrosis in the (t/t) and DAD(t/t) hearts. E, Transmission electron micrographs of sarcomeres show loss of the normal M-line structures in both cMyBP-C(t/t) and cMyBP-CDAD/(t/t)hearts. For each line between 240-539 individual sarcomeres were scored. For the control hearts, WT(t/t), 544 sarcomeres were counted with 9% blindly scored as “abnormal.” For the SAS/KO or ADA/KO animals, 15-18% had abnormal M lines, while for the DAD/KO animals, 244 sarcomeres were counted with 83% scored as having abnormal M-lines.

Sakthivel Sadayappan, et al. Circ Res. ;109(2):141-150.
3.
Figure 5

Figure 5. cMyBP-C domains and kinases that target the three-phosphorylation motifs. From: A Critical Function for Ser-282 in Cardiac Myosin Binding Protein-C Phosphorylation and Cardiac Function.

cMyBP-C is the 140-kDa thick filament sarcomeric proteins that consists of 11 domains; 8 immunoglobulin (oval) and 3 fibronectin type III (square) domains. The C0-domain, phosphorylation motif (m) and a cardiac-unique insertion in the C5-domain are unique (light green) to cMyBP-C, when compared to skeletal MyBP-C MyBP-C. The Proline/Alanine rich region (Pro-Ala) and hypothesized actin and myosin binding sites are marked. The three-phosphorylation motifs, Ser-282, Ser-273 and Ser-302 are shown (reference to the mouse identification number), which are targeted by protein kinase A (PKA),21 protein kinase C (PKC),21 Ca2+-calmodulin-activated kinase II (CaMKII),19 protein kinase D (PKD)22 and the 90kDa ribosomal s6 kinase (RSK).23 Amino acid sequences are numbered using Uniprot and NCBI reference numbers: note that Ser-282 in the mouse corresponds to Ser-284 in the human sequence.

Sakthivel Sadayappan, et al. Circ Res. ;109(2):141-150.
4.
Figure 2

Figure 2. Transgenic expression of cMyBP-C mutants in the cMyBP-C(t/t) null background. From: A Critical Function for Ser-282 in Cardiac Myosin Binding Protein-C Phosphorylation and Cardiac Function.

A, Schematic diagram of cMyBP-C illustrates domain structure and regions that interact with the other filament systems in the sarcomere. B, Mutations in the cMyBP-C transgenes. C, SDS-PAGE analysis of myofibrillar proteins from non-transgenic (NTG), cMyBP-C null (t/t), TG cMyBP-CWT (WT), cMyBP-CSAS (SAS) cMyBP-CADA (ADA) and cMyBP-CDAD (DAD) hearts. D, Expression levels of total cMyBP-C and the presence of myc-tagged (TG) cMyBP-C in the same mice were confirmed by Western blot analysis. E, Western blot analyses using phospho-specific antibodies on the in vitro dephosphorylated and phosphorylated NTG, cMyBP-CWT and cMyBP-CSAS myofibrils with phosphatase (control), PKCε, PKA and CaMKIIδ treatment. PKA phosphorylates all three sites, while PKC phosphorylates Ser-273 and Ser-302. CaMKIIδ was used at two different Ca2+ concentrations. F, Quantitative analysis of Western blot data (panel E). No significant differences were found between NTG and WT(t/t) groups. Values represent means ± S.E for each group (n=3), and designations denote significant differences (P<0.001) from WT(t/t) (* and #) controls.

Sakthivel Sadayappan, et al. Circ Res. ;109(2):141-150.
5.
Figure 1

Figure 1. Differential substrate specificities and regulation of Ser-273, Ser-282 and Ser-302 phosphorylation. From: A Critical Function for Ser-282 in Cardiac Myosin Binding Protein-C Phosphorylation and Cardiac Function.

A, Shown is the His6-tagged C1-M-C2 recombinant human cMyBP-C peptide that includes the C1-M-C2 domains with the 3 phosphorylation sites. In the normal, endogenous protein, each of these 3 serines is phosphorylatable. B, The ability of each residue to be phosphorylated by PKA, CaMKIIδ or PKCε is shown in Panels B and C, respectively. A dash indicates that the endogenous fragment with all 3 serines intact is present, and immunoblotting is indicated by “IB:” The serine (S) to alanine (A) mutation at each residue is indicated as Ser to Ala. Phosphorylation of each residue with each enzyme was assayed using our phospho-residue-specific antibodies via Western blot analyses. Ponceau-S staining of the membranes confirmed equal amounts of protein were loaded. D, E, Western blot analyses, using site-specific phospho cMyBP-C antibodies, of neonatal rat cardiomyocytes infected with constitutively active (CA) and dominant negative (DN) CaMKIIδ and (F, G) PKCε adenoviruses (Ad) at an MOI of 100 (n=5). C; negative control using an empty vector for infection, ISO; 50 nM isoproterenol treated (ISO) neonatal rat cardiomyocytes. H, I, At different time points after transverse aortic constriction (TAC) in mice, Western blot analysis of total heart proteins, using site-specific phospho cMyBP-C antibodies demonstrated that phosphorylation increased significantly after 30 minutes, but that Ser-282 phosphorylation was significantly reduced after 24 hours compared to Ser-273 and Ser-302 sites (n=6). *P<0.01 versus control or sham (n=5).

Sakthivel Sadayappan, et al. Circ Res. ;109(2):141-150.

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