The organization of myofilaments in cardiac myocytes and the phenotypes of myocardial cells are responsive to stresses exerted on the cell. By means of a cytoskeletal network of intermediate filaments (IF), stress and strain are transmitted to and from the extracellular matrix (ECM) via protein complexes of the costameres and the Z-discs of the sarcomeres. We have shown that the IF, synemin, which forms heteropolymers with desmin or vimentin, is also an A-kinase anchoring protein (AKAP). AKAPs regulate cAMP-dependent protein kinase (PKA) distribution in cells by binding with high affinity to the regulatory subunit (R), especially RII, of PKA, thus targeting PKA close to its substrate. Most AKAPs are multifunctional scaffolding proteins, acting as nodes of integration for several signaling pathways. We identified a binding site for RII on the synemin C-terminal tail, a region of synemin, which cross-links IFs, with the Z-disc, and with the costamere and intracellular organelles. Phosphoylation of IFs and IF associated proteins (IFAPs) regulate polymerization of IFs. This phosphorylation provides strong support for the idea that IFs constitute intrinsic components of signaling pathways. We hypothesize that synemin-anchored PKA regulates phosphorylation of myofibrillar and cytoskeletal substrates, which in turn regulates cytoskeletal and myofilament organization in the heart during contraction and during cardiac hypertrophy. These hypotheses will be tested in 3 Specific Aims:
In Specific Aim 1, we will determine the role of synemin as a scaffolding protein by (i) identifying the components of the macromolecular complex of synemin binding proteins and (ii) investigating the dynamic regulation of the complex. This will be performed by gel-free proteomic analysis of immunoprecipitated synemin binding proteins.
In Specific Aim 2, we will determine the role of synemin in targeting PKA in order to phosphoylate adjacent PKA substrates. Experiments will be carried out in neonatal and adult cardiac myocytes expressing the prolinated derivative of synemin, (synemin-P), which does not bind RII. We will then determine the effect of loss of PKA targeting on substrate phosphoylation.
In Specific Aim 3, we will examine the role of synemin targeted PKA in isolated neonatal and adult rat cardiac myocytes and in rat heart in vivo. We will (i) compare cardiac myocytes phenotype (upon synemin knockdown by RNAi, or by synemin-P expression by adenoviral gene transfer), then evaluate contractile response (ii) in isolated myocytes and (iii) in rat hearts in vivo after adenoviral gene transfer of synemin-P. Overall, these studies will provide new insights into the roles of synemin-targeted PKA in regulating cytoskeletal/myofibrillar substrate phosphoylation and contractile response during physiological and hypertrophic stress.
| García-Pelagio, Karla P; Muriel, Joaquin; O'Neill, Andrea et al. (2015) Myopathic changes in murine skeletal muscle lacking synemin. Am J Physiol Cell Physiol 308:C448-62 |
| Shanks, Maureen O; Lund, Linda M; Manni, Sabrina et al. (2012) Chromodomain helicase binding protein 8 (Chd8) is a novel A-kinase anchoring protein expressed during rat cardiac development. PLoS One 7:e46316 |
| Mauban, J R H; O'Donnell, M; Warrier, S et al. (2009) AKAP-scaffolding proteins and regulation of cardiac physiology. Physiology (Bethesda) 24:78-87 |
| Manni, Sabrina; Mauban, Joseph H; Ward, Christopher W et al. (2008) Phosphorylation of the cAMP-dependent protein kinase (PKA) regulatory subunit modulates PKA-AKAP interaction, substrate phosphorylation, and calcium signaling in cardiac cells. J Biol Chem 283:24145-54 |
