Plectin in skeletal muscle

Plectin is the prototype of intermediate filament (IF)-based cytolinker proteins. It strengthens cells mechanically by interlinking and anchoring cytoskeletal filaments, while as a scaffolding and docking platform for signaling proteins it controls cytoskeleton dynamics. The most common disease caused by plectin deficiency, epidermolysis bullosa (EB)-MD, is characterized by severe skin blistering and muscular dystrophy. Our previous studies on skeletal muscle suggest that four plectin isoforms are crucial for the integrity of myofibers by specifically targeting and linking desmin IF networks to Z-disks, costameres, mitochondria, and the nuclear/ER membrane system, with plectin deficiency leading to IF aggregation and mitochondrial dysfunction. The major new objective of this proposal is to mechanistically define plectin functions that go beyond integration and mechanical stabilization of muscle fibers. For this, we will assess whether plectin as a costameric signaling platform plays a role in mechanotransduction, study its role in regeneration of skeletal muscle, and address its function in mitochondrial fusion/fission and cytoskeleton-linked stress response. The experimental approach will rely on mostly already available plectin KO mouse models, myocyte cell cultures and intact whole muscles derived from these mice. The combined genetic, biochemical, cellular, molecular, and physiological approaches proposed are anticipated to yield a deeper understanding of how the skeletal muscle cytoskeleton copes with stress under normal conditions, after damage, and in disease. Finally, we consider this project as important for the development of future therapies for muscular dystrophies related to plectin and cytolinker proteins in general.

Plectin is the prototype of intermediate filament (IF)-based cytolinker proteins. It strengthens cells mechanically by interlinking and anchoring cytoskeletal filaments, and it serves as a scaffolding and docking platform for signaling proteins controlling cytoskeleton dynamics. The most common disease caused by plectin deficiency, epidermolysis bullosa simplex (EBS)-MD, is characterized by severe skin blistering and muscular dystrophy. Our previous studies on skeletal muscle suggested that plectin isoforms are crucial for the structural integrity of myofibers, with plectin deficiency leading to IF aggregation and mitochondrial dysfunction. The new objective of this project was to mechanistically define plectin functions that go beyond integration and mechanical stabilization of muscle fibers. In one of two main approaches, we assessed whether plectin, as a plasma membrane-associated (costameric) signaling platform, plays a role in metabolic processes and in mechanotransduction. By genetically altering mdx mice, an animal model for Duchenne muscular dystrophy, we could show that P1f, the costamere-associated isoform of plectin, acts as a local antagonist of microtubule (MT) network formation and thereby hinders vesicle-mediated MT-dependent transport processes, including glucose uptake. This is the first time, that a cytolinker protein has been shown to play a role in metabolic processes governing basic functions of muscle fibers. In studying plectin`s involvement in mechanotransduction, we showed P1f to be essential for the formation of a continuous axis between the extracellular matrix and the cell nucleus via the transmembrane integrin protein complex and the IF network system. Using fibroblast and myocyte cell cultures, we found that interruption of this axis, such as upon plectin deficiency, leads to interference with signal cascades controlling shape, polarization, migration, and adhesion of cells. Our results thus clearly established an important role of plectin in cellular signaling and mechanosensing of myofibers and related cell types. The focus in a second approach was on plectin`s role in regeneration of skeletal muscle fibers. Using a newly generated conditional plectin knockout mouse line (Pax7-Cre/cKO) specifically designed to suppress plectin expression is muscle precursor and regeneration-sustaining satellite cells, we found mutant mice to have a ~50% reduction of body weight and lifespan combined with kyphosis and massive muscle loss pointing towards developmental and skeletal muscle regeneration defects. Furthermore we found faster cell cycle rates of plectin- negative precursor cells suggesting that in mutant mice the pool of precursor/satellite cells was likely to be exhausted earlier, leading to fibrosis and muscle wasting. Additional features of the mutant mice were a hypoglycaemic condition, switch of muscle fiber types, altered mitochondrial activities, intracellular glycogen aggregates, and activation of signaling pathways known to decrease energy expending processes like protein synthesis. Our studies established for the first time at the organismic level an essential role of plectin in regulating signaling pathways involved in development, cell metabolism, and regeneration with direct implications for pathological conditions. Including additional studies with Pax7-Cre/cKO mice revealing a novel role of plectin in the structural integrity of the neuromuscular synapse, we expect ~15 original papers (fully or partially supported) and several review/perspective articles as outcome from this project.