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Thrombospondins (Thbs) are a family of five secreted matricellular glycoproteins in vertebrates that broadly affect cell-matrix interaction. While Thbs4 is known to protect striated muscle from disease by enhancing sarcolemmal stability through increased integrin and dystroglycan attachment complexes, here we show that Thbs3 antithetically promotes sarcolemmal destabilization by reducing integrin function, augmenting disease-induced decompensation. Deletion of Thbs3 in mice enhances integrin membrane expression and membrane stability, protecting the heart from disease stimuli. Transgene-mediated overexpression of α7β1D integrin in the heart ameliorates the disease predisposing effects of Thbs3 by augmenting sarcolemmal stability. Mechanistically, we show that mutating Thbs3 to contain the conserved RGD integrin binding domain normally found in Thbs4 and Thbs5 now rescues the defective expression of integrins on the sarcolemma. Thus, Thbs proteins mediate the intracellular processing of integrin plasma membrane attachment complexes to regulate the dynamics of cellular remodeling and membrane stability. Thrombospondin 4 has been shown to protect the heart and the skeletal muscle by enhancing matrix secretion and membrane stability thanks to its intracellular function. Here the authors show that thrombospondin 3 exacerbates injury-induced cardiomyopathy and promotes destabilization of the cardiomyocyte membrane by impairing integrin trafficking to the sarcolemma.

Skeletal muscle is highly sensitive to mutations in genes that participate in membrane stability and cellular attachment, which often leads to muscular dystrophy. Here we show that Thrombospondin-4 (Thbs4) regulates skeletal muscle integrity and its susceptibility to muscular dystrophy through organization of membrane attachment complexes. Loss of the Thbs4 gene causes spontaneous dystrophic changes with aging and accelerates disease in 2 mouse models of muscular dystrophy, while overexpression of mouse Thbs4 is protective and mitigates dystrophic disease. In the myofiber, Thbs4 selectively enhances vesicular trafficking of dystrophin-glycoprotein and integrin attachment complexes to stabilize the sarcolemma. In agreement, muscle-specific overexpression of Drosophila Tsp or mouse Thbs4 rescues a Drosophila model of muscular dystrophy with augmented membrane residence of βPS integrin. This functional conservation emphasizes the fundamental importance of Thbs’ as regulators of cellular attachment and membrane stability and identifies Thbs4 as a potential therapeutic target for muscular dystrophy. Muscle cells, also known as myofibers, need to be robust in order to withstand the physical stresses of contracting and relaxing. As a result, the cell surface membrane that surrounds myofibers is more strongly anchored to its surroundings than that of other cells. Muscular dystrophies are a group of muscle-wasting disorders that usually arise when this surface membrane becomes less stable. For example, mutations that affect a protein called dystrophin-glycoprotein or integrin protein complexes can cause muscular dystrophy since these proteins normally keep the membrane anchored and stable when the muscle contracts and relaxes. When myofibers in mammals become injured, as is the case during muscular dystrophy, they produce more proteins called thrombospondins – with thrombospondin-4 being the most common. However, until now it was not clear what these proteins did in muscle cells. Vanhoutte et al. hypothesized that thrombospondin-4 may protect injured myofibers and tested their theory by first deleting the gene for thrombospondin-4 from mutant mice that were predisposed to develop muscular dystrophy. This worsened the muscle wasting in the mutant mice, and furthermore, deleting the gene for thrombospondin-4 also caused otherwise normal mice to develop muscular dystrophy in their old age. Conversely, when Vanhoutte et al. artificially increased the levels of thrombospondin-4 in the myofibers, it protected the mice against muscular dystrophy. Additional experiments conducted in fruit flies demonstrated that the protective effects of thrombospondin are conserved or similar in insects too. Lastly, biochemical experiments in mouse and rat cells showed that thrombospondin-4 aids dystrophin-glycoproteins and integrins in getting to the cell surface membrane, increasing its stability. Overall these findings provide a clearer picture of the molecular underpinnings of muscular dystrophies. In the future, more experiments will have to focus on exactly how thrombospondins stabilize and direct dystrophin-glycoproteins and integrins to the cell surface membrane.

Efficient intracellular Ca²⁺ ([Ca²⁺]i) homeostasis in skeletal muscle requires intact triad junctional complexes comprised of t-tubule invaginations of plasma membrane and terminal cisternae of sarcoplasmic reticulum. Bin1 consists of a specialized BAR domain that is associated with t-tubule development in skeletal muscle and involved in tethering the dihydropyridine receptors (DHPR) to the t-tubule. Here, we show that Bin1 is important for Ca²⁺ homeostasis in adult skeletal muscle. Since systemic ablation of Bin1 in mice results in postnatal lethality, in vivo electroporation mediated transfection method was used to deliver RFP-tagged plasmid that produced short -hairpin (sh)RNA targeting Bin1 (shRNA-Bin1) to study the effect of Bin1 knockdown in adult mouse FDB skeletal muscle. Upon confirming the reduction of endogenous Bin1 expression, we showed that shRNA-Bin1 muscle displayed swollen t-tubule structures, indicating that Bin1 is required for the maintenance of intact membrane structure in adult skeletal muscle. Reduced Bin1 expression led to disruption of t-tubule structure that was linked with alterations to intracellular Ca²⁺ release. Voltage-induced Ca²⁺ released in isolated single muscle fibers of shRNA-Bin1 showed that both the mean amplitude of Ca²⁺ current and SR Ca²⁺ transient were reduced when compared to the shRNA-control, indicating compromised coupling between DHPR and ryanodine receptor 1. The mean frequency of osmotic stress induced Ca²⁺ sparks was reduced in shRNA-Bin1, indicating compromised DHPR activation. ShRNA-Bin1 fibers also displayed reduced Ca²⁺ sparks' amplitude that was attributed to decreased total Ca²⁺ stores in the shRNA-Bin1 fibers. Human mutation of Bin1 is associated with centronuclear myopathy and SH3 domain of Bin1 is important for sarcomeric protein organization in skeletal muscle. Our study showing the importance of Bin1 in the maintenance of intact t-tubule structure and ([Ca²⁺]i) homeostasis in adult skeletal muscle could provide mechanistic insight on the potential role of Bin1 in skeletal muscle contractility and pathology of myopathy.

Efficient intracellular Ca.sup.2+ ([Ca.sup.2+ ]i) homeostasis in skeletal muscle requires intact triad junctional complexes comprised of t-tubule invaginations of plasma membrane and terminal cisternae of sarcoplasmic reticulum. Bin1 consists of a specialized BAR domain that is associated with t-tubule development in skeletal muscle and involved in tethering the dihydropyridine receptors (DHPR) to the t-tubule. Here, we show that Bin1 is important for Ca.sup.2+ homeostasis in adult skeletal muscle. Since systemic ablation of Bin1 in mice results in postnatal lethality, in vivo electroporation mediated transfection method was used to deliver RFP-tagged plasmid that produced short -hairpin (sh)RNA targeting Bin1 (shRNA-Bin1) to study the effect of Bin1 knockdown in adult mouse FDB skeletal muscle. Upon confirming the reduction of endogenous Bin1 expression, we showed that shRNA-Bin1 muscle displayed swollen t-tubule structures, indicating that Bin1 is required for the maintenance of intact membrane structure in adult skeletal muscle. Reduced Bin1 expression led to disruption of t-tubule structure that was linked with alterations to intracellular Ca.sup.2+ release. Voltage-induced Ca.sup.2+ released in isolated single muscle fibers of shRNA-Bin1 showed that both the mean amplitude of Ca.sup.2+ current and SR Ca.sup.2+ transient were reduced when compared to the shRNA-control, indicating compromised coupling between DHPR and ryanodine receptor 1. The mean frequency of osmotic stress induced Ca.sup.2+ sparks was reduced in shRNA-Bin1, indicating compromised DHPR activation. ShRNA-Bin1 fibers also displayed reduced Ca.sup.2+ sparks' amplitude that was attributed to decreased total Ca.sup.2+ stores in the shRNA-Bin1 fibers. Human mutation of Bin1 is associated with centronuclear myopathy and SH3 domain of Bin1 is important for sarcomeric protein organization in skeletal muscle. Our study showing the importance of Bin1 in the maintenance of intact t-tubule structure and ([Ca.sup.2+ ]i) homeostasis in adult skeletal muscle could provide mechanistic insight on the potential role of Bin1 in skeletal muscle contractility and pathology of myopathy.