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Intermediate filaments (IFs) in cardiomyocytes consist primarily of desmin, surround myofibrils at Z disks, and transmit forces from the contracting myofilaments to the cell surface through costameres at the sarcolemma and desmosomes at intercalated disks. Synemin is a type IV IF protein that forms filaments with desmin and also binds α-actinin and vinculin. Here we examine the roles and expression of the α and β forms of synemin in developing rat cardiomyocytes. Quantitative PCR showed low levels of expression for both synemin mRNAs, which peaked at postnatal day 7. Synemin was concentrated at sites of cell--cell adhesion and at Z disks in neonatal cardiomyocytes. Overexpression of the individual isoforms showed that α-synemin preferentially localized to cell-cell junctions, whereas β-synemin was primarily at the level of Z disks. An siRNA targeted to both synemin isoforms reduced protein expression in cardiomyocytes by 70% and resulted in a failure of desmin to align with Z disks and disrupted cell--cell junctions, with no effect on sarcomeric organization. Solubility assays showed that α-synemin was soluble and interacted with sarcomeric-actinin by coimmunoprecipitation, while α-synemin and desmin were insoluble. We conclude that β-synemin mediates the association of desmin IFs with Z disks, whereas-synemin stabilizes junctional complexes between cardiomyocytes. [ABSTRACT FROM AUTHOR]

Synemin binds PKA RII via blot overlays and co-immunoprecipitates with RII from adult rat cardiomyocytes (CM) through an amphipathic helical AKAP domain. We also show colocalization of synemin with RII at Z-lines and the perinuclear region using immunohistochemistry with adult CM. In isolated rat neonatal CMs, synemin localized to the sarcolemma but not to filamentous, cytoplasmic desmin. There are two known isoforms of synemin in rat (H and M) that differ by 932bp in the C-terminus. Using antibodies to each isoform, the two differ in their subcellular location: H is at the sarcolemma, while M is at striations. A polyclonal antibody against H recognizes 210kDa and 100kDa bands (SDS-PAGE weights) via western blots of CM lysates, suggesting a new putative isoform. Studies by other labs demonstrated an L isoform comprising the rod domain (exons 1-3) and a truncated tail (exon 5) in mouse brain, skeletal muscle, and heart that may be expressed in rat neonatal or adult CMs. Using affinity-tagged constructs, we are isolating the multimolecular protein complexes formed by each isoform. We hypothesize that these isoforms have variable subcellular locations and binding partners and may be temporally regulated. We conclude that synemin isoforms have different functions and target PKA near different substrates within myocytes. [ABSTRACT FROM AUTHOR]