Robert J. Bloch

Erratum to: Differential distribution of dystrophin and β-spectrin at the sarcolemma of fast twitch skeletal muscle fibers

We used double label immunofluorescence and confocal microscopy to examine the organization of β-spectrin and dystrophin at the sarcolemma of fast twitch myofibers in the Extensor Digitorum Longus (EDL) of the rat. Both β-spectrin and dystrophin are concentrated in costameres, a rectilinear sarcolemmal array composed of longitudinal strands and transverse elements overlying Z and M lines. In contrast, intercostameric regions, lying between these linear structures, contain significant levels of dystrophin but little detectable β-spectrin. The dystrophin-associated proteins, syntrophin and β-dystroglycan, are also concentrated at costameres but, like dystrophin, are present in intercostameric regions as well. Dystrophin is present at costameres and intercostameric regions in fast twitch muscles of the mouse but is absent from all regions of the sarcolemma in the mdx mouse, which lacks dystrophin. Areas of the sarcolemma near myonuclei also contain dystrophin without β-spectrin, consistent with the idea that the distribution of dystrophin at the sarcolemma is not dependent on β-spectrin. We conclude that dystrophin is present under all areas of the sarcolemma. The increased fragility of the sarcolemma in patients with Duchennes muscular dystrophy may be explained in part by the absence of dystrophin not only from costameres, but also from intercostameric regions.

Association of small ankyrin 1 with the sarcoplasmic reticulum

Small ankyrin 1, or sAnk1, is a small, alternatively spliced product of the erythroid ankyrin gene, ANK1, that is expressed in striated muscle and concentrated in the network sarcoplasmic reticulum (SR) surrounding the Z disks and M lines. We have characterized sAnk1 in muscle homogenates and SR vesicles, and have identified the region that targets it to the network SR. Selective extractions and partitioning into Triton X-114 show that sAnk1 behaves like the SR Ca-ATPase and so is an integral protein of the SR membrane. Mild proteolytic treatment of isolated SR vesicles indicates that sAnk1 is oriented with its hydrophilic, C-terminal sequence exposed to the solution, which is equivalent to the cytoplasmic face of the SR membrane in situ. SDS-PAGE in non-reducing gels suggests that sAnk1 is present as dimers and larger oligomers in the native SR. These results suggest that sAnk1 is oligomeric and oriented with its C-terminus exposed to the cytoplasm, where it may interact with proteins of the contractile apparatus. The N-terminal 29 amino acid hydrophobic sequence of sAnk1, which is predicted to span the SR membrane, is sufficient to target proteins to and anchor them in internal membranes of HEK 293 cells. It also targets reporter proteins to the network SR of skeletal myofibers and is thus the first example of a sequence that targets proteins to a particular compartment of the SR.

Postnatal changes in sarcolemmal organization in the mdx mouse

The tibialis anterior muscles of mdx mice degenerate between 3 and 4 weeks after birth and then partially recover. We show that the membrane cytoskeleton at the mdx sarcolemma is disorganized at 18-days postnatal, and becomes more disorganized at 4 weeks compared to earlier or later times. Mdx muscle at 18 days have few central nuclei, suggesting that it has not yet sustained significant damage. The variance of myofiber diameter confirms that the mdx pathology is greatest at 4 weeks, when the sarcolemma is most disorganized. Sarcolemmal disorganization in the mdx does not involve contractile structures, nor is it seen in age-matched controls. In revertant mdx fibers expressing dystrophin, sarcolemmal organization is similar to controls. Our results suggest that the absence of dystrophin results in the disorganization of the sarcolemma, even in 18-day-old mice, and that the extent of disorganization is greater when the myopathy is most severe.

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.

Elastic Properties of the Sarcolemma‐Costamere Complex of Muscle Cells in Normal Mice

Costameres at the sarcolemma of skeletal myofibers are thought to mediate the lateral transmission of force from the myofibrils to the extracellular matrix. We applied elastimetry to single myofibers from the Extensor digitorum longus muscles of mice to measure the biomechanical properties of the sarcolemma and the costameres as a function of sarcomere length. Suction pressure (P) applied by the elastimeter to the sarcolemma generated a bleb of variable height, which depended on P and sarcomere length. Connections between the sarcolemma and nearby myofibrils broke as P increased. Pressure, tension, force and stiffness (k) were calculated using the displacement pressure curves. With Laplace’s and Hooke’s equations, we estimated tensions generated by the sarcolemma attached to the myofibrils (Tc+s) and the sarcolemma after detachment (Ts). Tc+s − Ts = Tc, the maximal tension sustained by the costameres. The sarcolemma‐costamere complex was represented as a lumped elastic model, in which the elasticity of ea...

Absence of synemin causes hypertrophy in murine heart

Heart disease is the most frequent cause of death in the world and becomes more common with aging. Cardiomyopathies have been linked to changes in structural proteins, including intermediate filament (IF). IFs associate with the contractile machinery and costameres of striated muscle and with the 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 acts as an A-kinase anchoring protein (AKAP). We used echocardiography, and immunofluorescence labeling to obtain the results. Here, we report that synemin-null (synm -/-) cardiac muscle shows left ventricular remodeling, contractile dysfunction, hypertrophy, and an increment of total body weight suggesting a mixed cardiomyopathy (dilated and hypertrophic) with a profound dilated phenotype. Data suggest that synemin plays an important regulatory role in the heart and that the consequences of its absence are profound.

Characterization of skeletal muscle in the synemin knock-out mouse

Diseases linked to intermediate filament (IF) proteins are associated with defects in the organization of the contractile apparatus of skeletal and cardiac muscle and its links to costameres, which connect the sarcomeres to the cell membrane. Synemin is a large IF protein that associates with dystrobrevin, vinculin, and talin at costameres of the cell membrane of striated muscle, as well as with α-actinin and desmin at the Z disks. Synemin can be expressed in either 210 kDa α- or 180 kDa β- alternatively spliced forms. We generated mice null for synemin by homologous recombination to study synemins function in skeletal muscle. Skeletal muscle in the knock out (syn KO) mouse does not make synemin mRNA or protein. Preliminary characterization of the syn KO mouse suggests that it has a mild skeletal muscle phenotype. The organization of costameres appears to be normal. Treadmill running uphill test results was not significantly affected when compared to controls at any age. More notably, the biomechanical p...

Passive Viscoelastic Properties of Costameres in EDL Skeletal Muscle in Normal and Dystrophin Null Mice

Costameres at the sarcolemmal skeletal myofibers transmit the lateral force generated by myofibrils from them to the extracellular matrix. We used an elastimeter method by which sucking pressure is applied through a micropipette to the surface membrane of single mice myofibers of the Extensor digitorum longus to measure the viscoelasticity of the sarcolemma‐costamere complex as a function of sarcomere length (SL). Constant suction pressure applied to the sarcolemma generated a sarcolemmal‐costamere‐myofibril bleb of variable height depending on the sucking pressure and SL. It took some time for the bleb to reach a stable height after applying the pressure. This time delay indicates that the sarcolemma‐costamere‐myofibril system acts as a viscoelastic system. We undertook the present experiments to measure the height stabilization time of the bleb at different sarcomere lengths from which we estimated the viscoelastic parameters of the system. The time course of the bleb formation was biphasic and reached ...

Modeling the Cell Muscle Membrane from Normal and Desmin‐ or Dystrophin‐null Mice as an Elastic System

Two of the most important proteins linking the contractile apparatus and costameres at the sarcolemma of skeletal muscle fibers are dystrophin and desmin. We have developed an elastic model of the proteins that link the sarcolemma to the myofibrils. This is a distributed model, with an elastic constant, k, that includes the main protein components of the costameres. The distributed spring model is composed of parallel units attached in series. To test the model, we performed experiments in which we applied negative pressure, generated by an elastimeter, to a small area of the sarcolemma from single myofiber. The negative pressure formed a bleb of variable height, dependent on the pressure applied. We normalized our measurements of k in dystrophin‐null (mdx) and desmin‐null (des‐/‐) mice to the value we obtained for wild type (WT) mice, which was set at 1.0. The relative experimental value for the stiffness of myofibers from mice lacking dystrophin or desmin was 0.5 and 0.7, respectively. The theoretical k values of the individual elements were obtained using neural networks (NN), in which the input was the k value for each parallel spring component and the output was the solution of each resulting parallel system. We compare the experimental values of k in control and mutant muscles to the theoretical values obtained by NN for each protein. Computed theoretical values were 0.4 and 0.8 for dystrophin‐ and desmin‐null muscles, respectively, and 0.9 for WT, in reasonable agreement with our experimental results. This suggests that, although it is a simplified spring model solved by NN, it provides a good approximation of the distribution of spring elements and the elastic constants of the proteins that form the costameres. Our results show that dystrophin is the protein that contributes more than any other to the strength of the connections between the sarcolemma and the contractile apparatus, the costameres.Two of the most important proteins linking the contractile apparatus and costameres at the sarcolemma of skeletal muscle fibers are dystrophin and desmin. We have developed an elastic model of the proteins that link the sarcolemma to the myofibrils. This is a distributed model, with an elastic constant, k, that includes the main protein components of the costameres. The distributed spring model is composed of parallel units attached in series. To test the model, we performed experiments in which we applied negative pressure, generated by an elastimeter, to a small area of the sarcolemma from single myofiber. The negative pressure formed a bleb of variable height, dependent on the pressure applied. We normalized our measurements of k in dystrophin‐null (mdx) and desmin‐null (des‐/‐) mice to the value we obtained for wild type (WT) mice, which was set at 1.0. The relative experimental value for the stiffness of myofibers from mice lacking dystrophin or desmin was 0.5 and 0.7, respectively. The theoretical k...