Knockout mice and cell models for muscular plectinopathies

The most common disease caused by plectin deficiency, epidermolysis bullosa (EB)-MD, is characterized by muscular dystrophy and severe skin blistering. EB-MD patients and plectin-deficient mice display massive desmin aggregation, the hallmark of MFM. Our previous studies have shown that the 4 major plectin isoforms expressed in muscle are crucial for the integrity of myofibers by specifically targeting and anchoring desmin IF networks to Z- disks, costameres, mitochondria, and the nuclear/SR membrane system. One of the major achievements of our ongoing project was the establishment of immortalized plectin-deficient myocyte cell lines that spontaneously develop desmin aggregates when differentiated into contractile myotubes. We will take advantage of these systems and of an ample collection of conditional (MCK-Cre/Pax7-Cre) and muscle isoform-specific plectin knockout mouse lines to address the following new objectives: i) analysis of the molecular pathogenesis of plectin-related MFMs with focus on mechanisms involved in desmin network collapse, isoform-specific interaction partners, and neuromuscular end plate dysfunction; ii) assessment of novel treatment concepts through phenotype rescue (removal of protein aggregates and gene transfer); and iii) analysis of biomechanical properties of plectin-deficient myocytes and muscle fibers.

The most common disease caused by plectin deficiency, epidermolysis bullosa (EB)-MD, is characterized by muscular dystrophy and severe skin blistering. EB-MD patients and plectin-deficient mice display massive protein aggregation in their skeletal muscle cells, the hallmark of myofibrillary myopathies (MFMs). We have established cell culture systems mimicking the pathological hallmarks of MFMs and took advantage of our ample collection of conditional and muscle isoform-specific plectin knockout mouse lines to address the following objectives: (i) Gain a better understanding of the pathogenesis underlying plectinopathies by unraveling the mechanisms of plectin/desmin networking, including the contractile apparatus, the neuromuscular synapse, nuclei, and mitochondria, ii) develop pharmacological treatment and/or gene transfer concepts for the removal of protein aggregates from MFM myofibers, and iii) analyze biomechanical properties and the mechanotransduction potential of plectin-deficient cells and tissues.In the context of objective (i) our studies revealed that one of the plectin isoforms, P1, by recruiting desmin IFs to myonuclei affects nuclear morphology and gene expression, and thus plays an important role in mechanotransduction. Equally important roles were revealed for isoforms P1d and P1b in affecting the morphology and functionality of the contractile apparatus and mitochondria, while P1f could be shown to be of vital importance for neurosynapse integrity as well as glucose uptake. The outcome of objective (ii)-based studies was highly satisfying, as 4-PBA, a chemical chaperon already approved by the FDA for different indications, was found to greatly reduce or prevent the formation of protein aggregates in MFM cell cultures and intact muscles, combined with improved muscle performance of mice. This study provided the basis for ongoing initiatives for clinical trials with MFM patients. We also prepared and tested cDNA expression plasmids for plectin mini gene versions, setting the stage for gene therapeutic approaches with MFM mice and eventually human patients. Regarding objective (iii), we could show that dysfunctional plectin severely affects the stiffness, adhesion strength, and cytoskeletal dynamics of myocytes and other types of cells in cell type/isoform-specific manner. In a series of complementary studies, the dominant role of plectin in mechanotransduction could be confirmed and extended to other cell systems, including vascular endothelial cells, fibroblasts, keratinocytes, and a number of cancer cell systems. In all, our results significantly advanced our understanding of the molecular mechanisms underlying plectinopathies and other MFMs, and opened a way for a possible treatment of these diseases. Without doubt this project greatly benefited from the joining of forces of several groups within the framework provided by the international research program Molecular Pathogenesis of Myofibrillar Myopathies of which it was part of.