Biomechanical Properties of the Sarcolemma and Costameres of Skeletal Muscle Lacking Desmin.

Intermediate filaments (IFs), composed primarily by desmin and keratins, link the myofibrils to each other, to intracellular organelles, and to the sarcolemma. There they may play an important role in transfer of contractile force from the Z-disks and M-lines of neighboring myofibrils to costameres at the membrane, across the membrane to the extracellular matrix, and ultimately to the tendon ("lateral force transmission"). We measured the elasticity of the sarcolemma and the connections it makes at costameres with the underlying contractile apparatus of individual fast twitch muscle fibers of desmin-null mice. By positioning a suction pipet to the surface of the sarcolemma and applying increasing pressure, we determined the pressure at which the sarcolemma separated from nearby sarcomeres, Pseparation, and the pressure at which the isolated sarcolemma burst, Pbursting. We also examined the time required for the intact sarcolemma-costamere-sarcomere complex to reach equilibrium at lower pressures. All measurements showed the desmin-null fibers to have slower equilibrium times and lower Pseparation and Pbursting than controls, suggesting that the sarcolemma and its costameric links to nearby contractile structures were weaker in the absence of desmin. Comparisons to earlier values determined for muscles lacking dystrophin or synemin suggest that the desmin-null phenotype is more stable than the former and less stable than the latter. Our results are consistent with the moderate myopathy seen in desmin-null muscles and support the idea that desmin contributes significantly to sarcolemmal stability and lateral force transmission.

Absence of synemin in mice causes structural and functional abnormalities in heart.

Cardiomyopathies have been linked to changes in structural proteins, including intermediate filament (IF) proteins located in the cytoskeleton. IFs associate with the contractile machinery and costameres of striated muscle and with intercalated disks in the heart. Synemin is a large IF protein that mediates the association of desmin with Z-disks and stabilizes intercalated disks. It also acts as an A-kinase anchoring protein (AKAP). In murine skeletal muscle, the absence of synemin causes a mild myopathy. Here, we report that the genetic silencing of synemin in mice (synm -/-) causes left ventricular systolic dysfunction at 3months and 12-16months of age, and left ventricular hypertrophy and dilatation at 12-16months of age. Isolated cardiomyocytes showed alterations in calcium handling that indicate defects intrinsic to the heart. Although contractile and costameric proteins remained unchanged in the old synm -/- hearts, we identified alterations in several signaling proteins (PKA-RII, ERK and p70S6K) critical to cardiomyocyte function. Our data suggest that synemin plays an important regulatory role in the heart and that the consequences of its absence are profound.

Effects of the plasmid-encoded toxin of enteroaggregative Escherichia coli on focal adhesion complexes.

Enteroaggregative Escherichia coli (EAEC) is an emerging diarrheal pathogen. Many EAEC strains produce the plasmid-encoded toxin (Pet), which exerts cytotoxic effects on human intestinal tissue. Pet-intoxicated HEp-2 cells exhibit rounding and detachment from the substratum, accompanied by loss of F-actin stress fibers and condensation of the spectrin-containing membrane cytoskeleton. Although studies suggest that Pet directly cleaves spectrin, it is not known whether this is the essential mode of action of the toxin. In addition, the effects of Pet on cytoskeletal elements other than actin and spectrin have not been reported. Here, we demonstrate by immunofluorescence that upon Pet intoxication, HEp-2 and HT29 cells lose focal adhesion complexes (FAC), a process that includes the redistribution of focal adhesion kinase (FAK), α-actinin, paxillin, vinculin, F-actin, and spectrin itself. This redistribution was coupled with the depletion of phosphotyrosine labeling at FACs. Immunoblotting and immunoprecipitation experiments revealed that FAK was tyrosine dephosphorylated, before the redistribution of FAK and spectrin. Moreover, phosphatase inhibition blocked cell retraction, suggesting that tyrosine dephosphorylation is an event that precedes FAK cleavage. Finally, we show that in vitro tyrosine-dephosphorylated FAK was susceptible to Pet cleavage. These data suggest that mechanisms other than spectrin redistribution occur during Pet intoxication.