Comparison of polysomal and nuclear poly(A)-containing RNA populations from normal rat liver and Novikoff hepatoma.

Polysomal and nuclear poly(A)-containing RNA of normal rat liver and Novikoff hepatoma cells have been compared by cDNA.RNA hybridization kinetics. Homologous hybridization reactions revealed at total kinetic complexity of about 1.6 X 10(10) and 1.38 X 10(10) daltons for liver and Novikoff mRNA respectively. The high abundance component present in liver cannot be detected in Novikoff. It was found from heterologous reactions that about 30% by weight of mRNA sequences are specific to liver. Determination of the nuclear poly(A)-containing RNA complexities revealed that about 5.5% and 4% of the haploid genome is expressed in the liver and Novikoff respectively. In a heterologous reaction, up to 30% of the liver cDNA failed to form hybrids with Novikoff nuclear RNA. Cross hybridizations have further revealed abundance shifts in both nuclear and polysomal RNA populations. Some sequences abundant in liver are less abundant in Novikoff and some rare liver sequences are relatively abundant in Novikoff.     

Caspase proteolysis of desmin produces a dominant-negative inhibitor of intermediate filaments and promotes apoptosis

Caspase cleavage of key cytoskeletal proteins, including several intermediate filament proteins, triggers the dramatic disassembly of the cytoskeleton that characterizes apoptosis. Here we describe the muscle-specific intermediate filament protein desmin as a novel caspase substrate. Desmin is cleaved selectively at a conserved Asp residue in its L1-L2 linker domain (VEMD downward arrow M(264)) by caspase-6 in vitro and in myogenic cells undergoing apoptosis. We demonstrate that caspase cleavage of desmin at Asp(263) has important functional consequences, including the production of an amino-terminal cleavage product, N-desmin, which is unable to assemble into intermediate filaments, instead forming large intracellular aggregates. Moreover, N-desmin functions as a dominant-negative inhibitor of filament assembly, both for desmin and the structurally related intermediate filament protein vimentin. We also show that stable expression of a caspase cleavage-resistant desmin D263E mutant partially protects cells from tumor necrosis factor-alpha-induced apoptosis. Taken together, these results indicate that caspase proteolysis of desmin at Asp(263) produces a dominant-negative inhibitor of intermediate filaments and actively participates in the execution of apoptosis. In addition, these findings provide further evidence that the intermediate filament cytoskeleton has been targeted systematically for degradation during apoptosis.     

Sarcolemmal organization in skeletal muscle lacking desmin: evidence for cytokeratins associated with the membrane skeleton at costameres

The sarcolemma of fast-twitch muscle is organized into "costameres," structures that are oriented transversely, over the Z and M lines of nearby myofibrils, and longitudinally, to form a rectilinear lattice. Here we examine the role of desmin, the major intermediate filament protein of muscle in organizing costameres. In control mouse muscle, desmin is enriched at the sarcolemmal domains that lie over nearby Z lines and that also contain beta-spectrin. In tibialis anterior muscle from mice lacking desmin due to homologous recombination, most costameres are lost. In myofibers from desmin -/- quadriceps, by contrast, most costameric structures are stable. Alternatively, Z line domains may be lost, whereas domains oriented longitudinally or lying over M lines are retained. Experiments with pan-specific antibodies to intermediate filament proteins and to cytokeratins suggest that control and desmin -/- muscles express similar levels of cytokeratins. Cytokeratins concentrate at the sarcolemma at all three domains of costameres when the latter are retained in desmin -/- muscle and redistribute with beta-spectrin at the sarcolemma when costameres are lost. Our results suggest that desmin associates with and selectively stabilizes the Z line domains of costameres, but that cytokeratins associate with all three domains of costameres, even in the absence of desmin.     

Desmin knockout muscles generate lower stress and are less vulnerable to injury compared with wild-type muscles

The functional roleof the skeletal muscle intermediate filament system was investigated bymeasuring the magnitude of muscle force loss after cyclic eccentriccontraction (EC) in normal and desmin null mouse extensor digitorumlongus muscles. Isometric stress generated was significantly greater inwild-type (313 ± 8 kPa) compared with knockout muscles (276 ± 13 kPa) before EC (P < 0.05), but 1 h after 10 ECs, both muscle types generated identical levels of stress (~250kPa), suggesting less injury to the knockout. Differences in injurysusceptibility were not explained by the different absolute stresslevels imposed on wild-type versus knockout muscles (determined bytesting older muscles) or by differences in fiber length or mechanicalenergy absorbed. Morphometric analysis of longitudinal electronmicrographs indicated that Z disks from knockout muscles were morestaggered (0.36 ± 0.03 µm) compared with wild-type muscles(0.22 ± 0.03 µm), which may indicate that the knockoutcytoskeleton is more compliant. These data demonstrate that lack of theintermediate filament system decreases isometric stress production andthat the desmin knockout muscle is less vulnerable to mechanical injury.

     

Altered expression of calreticulin during the development of fibrosis

Tissue damage following injury leads to inflammation and fibrosis. To understand the molecular mechanisms and the proteins involved in the fibrotic process, we used the well-established unilateral ureteric obstruction rat model and we analyzed the alterations at early and late time intervals using a classical proteomic approach. Data analysis demonstrates a correlation between calreticulin up-regulation and progression of fibrosis. Calreticulin is involved in Ca++ homeostasis but has not been previously implicated in animal models of fibrosis. Proteomic analysis consistently revealed up-regulation of calreticulin in both early and late time intervals. These findings were further confirmed by biochemical and morphological approaches. Next, animal models of lung fibrosis (bleomycin-induced) and heart fibrosis (desmin-null) were examined. In the lung model, calreticulin expression was up-regulated from early time intervals, whereas in the heart model no change in the expression of calreticulin was observed. In addition, TGF-beta, a well known major contributing factor in several fibrotic processes, was found to up-regulate calreticulin in cultured human proximal tubule epithelial cells. The above observations suggest that calreticulin might be involved in fibrotic processes; however the mechanism(s) underlying its possible involvement are yet unresolved.     

Desmin integrates the three-dimensional mechanical properties of muscles

Striated muscle is a linear motor whose properties have been defined in terms of uniaxial structures. The question addressed here is what contribution is made to the properties of this motor by extramyofilament cytoskeletal structures that are not aligned in parallel with the myofilaments. This question arose from observations that transverse loads increase muscle force production in diaphragm but not in the hindlimb muscle, thereby indicating the presence of structures that couple longitudinal and transverse properties of diaphragmatic muscle. Furthermore, we find that the diaphragms of null mutants for the cytoskeletal protein desmin show 1) significant reductions in coupling between the longitudinal and transverse properties, indicating for the first time a role for a specific protein in integrating the three-dimensional mechanical properties of muscle, 2) significant reductions in the stiffness and viscoelasticity of muscle, and 3) significant increases in tetanic force production. Thus desmin serves a complex mechanical function in diaphragm muscle by contributing both to passive stiffness and viscoelasticity and to modulation of active force production in a three-dimensional structural network. Our finding changes the paradigm of force transmission among cells by placing our understanding of the function of the cytoskeleton in the context of the structural and mechanical complexity of muscles.     

Paracrine promotion of cardiomyogenesis in embryoid bodies by LIF modulated endoderm

In the vertebrate embryo the heart is the first organ to form. Embryonic and extra-embryonic tissues are supposed to contribute to cardiac lineage commitment before and during gastrulation in a paracrine fashion. Evidence has accumulated that factors secreted by the anterior lateral endoderm and extra-embryonic endoderm contribute to cardiomyogenesis. Here we exploit in vitro differentiation of embryonic stem cells in embryoid bodies to study differentiation of the extraembryonic endodermal lineage, gastrulation-like processes, and the influence of endoderm on cardiomyogenesis. We demonstrate that in embryoid bodies primitive endoderm differentiates to visceral and parietal endoderm and that parietal endoderm influences onset of cardiomyogenesis in a concentration-dependent manner. Both increased concentrations of leukemia inhibitory factor and its absence in lif-/- embryoid bodies hampered parietal endoderm formation. Reduced differentiation of parietal endoderm correlated with an attenuation of cardiomyogenesis even in the presence of LIE These and previous results suggest that leukemia inhibitory factor is directly and indirectly, via endoderm formation, involved in the regulation of cardiomyogenesis. Increased proliferation of parietal endoderm in lifr -/- embryoid bodies and addition of conditioned lif -/- cell culture supernatant promoted cardiomyogenesis, demonstrating for the first time that parietal endoderm also contributes to cardiomyogenesis in embryoid bodies in a paracrine and leukemia inhibitory factor and its receptor independent pathway. New factors signaling independently of the leukemia inhibitory-factor receptor pathway may sustain cardiomyocyte cell proliferation and thus be a future target for gene therapy of cardiomyopathies and cell therapy of the myocardium.     

Μyospryn: a multifunctional desmin-associated protein

Desmin, the muscle-specific intermediate filament protein, forms a 3D scaffold that links the contractile apparatus to the costameres of plasma membrane, intercalated disks, the nucleus, and also other membranous organelles. The cellular scaffold formed by desmin and its binding partners might be implicated in signaling and trafficking processes, vital mechanisms for the survival of the mammalian cell. One novel desmin-associated protein is the tripartite motif-like protein myospryn. Myospryn was initially identified as an associated partner to the biogenesis of lysosome-related organelles complex 1 protein dysbindin, implicating its potential involvement in vesicle trafficking and organelle biogenesis and/or positioning. Myospryn is also an A kinase anchoring protein, raising the possibility that together with desmin and other cytoskeletal and signaling proteins, it could participate in the subcellular targeting of protein kinase A activity in striated muscle. As with desmin, different members of this scaffold might play a crucial role in the pathogenesis of muscle disease, since any disturbance in these highly coordinated signaling pathways is expected to compromise efficient maintenance of structure–function integrity of muscle and lead to different cardiac and skeletal myopathies.     

Desmin mediates TNF-alpha-induced aggregate formation and intercalated disk reorganization in heart failure

We explored the involvement of the muscle-specific intermediate filament protein desmin in the model of tumor necrosis factor alpha (TNF-alpha)-induced cardiomyopathy. We demonstrate that in mice overexpressing TNF-alpha in the heart (alpha-myosin heavy chain promoter-driven secretable TNF-alpha [MHCsTNF]), desmin is modified, loses its intercalated disk (ID) localization, and forms aggregates that colocalize with heat shock protein 25 and ubiquitin. Additionally, other ID proteins such as desmoplakin and beta-catenin show similar localization changes in a desmin-dependent fashion. To address underlying mechanisms, we examined whether desmin is a substrate for caspase-6 in vivo as well as the implications of desmin cleavage in MHCsTNF mice. We generated transgenic mice with cardiac-restricted expression of a desmin mutant (D263E) and proved that it is resistant to caspase cleavage in the MHCsTNF myocardium. The aggregates are diminished in these mice, and D263E desmin, desmoplakin, and beta-catenin largely retain their proper ID localization. Importantly, D263E desmin expression attenuated cardiomyocyte apoptosis, prevented left ventricular wall thinning, and improved the function of MHCsTNF hearts.