Investigating the Role of Lamins A/C in Maintaining Nuclear Stability Using a Novel 3D in vitro Model of Muscular Dystrophy

Introduction: : Striated muscle laminopathies are a group of diseases that cause muscle atrophy and fibrosis in cardiac and skeletal muscles leading to dilated cardiomyopathy or muscular dystrophy. The only effective treatment options that exist for striated muscle laminopathies are organ transplants, motivating the search for new therapeutic approaches. Laminopathies are caused by mutations in the LMNA gene, which codes for nuclear proteins lamins A/C that provide structure to the cell’s nucleus, protect it from mechanical forces, organize chromatin, and interact with various transcriptional regulators. LMNA mutations cause cells to have softer, more fragile nuclei that are prone to rupture. However, it is still unknown how nuclear fragility contributes to muscular dystrophy and to what extent LMNA mutations perturb muscle differentiation and function. To address these questions, we have developed a 3D in vitro model of Emery Dreifuss Muscular Dystrophy (EDMD) skeletal muscle, which recapitulates the impaired nuclear stability of diseased muscle tissue in vivo. In addition, our model enables us to study structural and signaling defects during myoblast differentiation and maturation through high-resolution imaging. Within this culture system, mechanical forces from neighboring cells and the extracellular matrix promote the formation of elongated myotubes and early myofibers. Traditional 2D in vitro culture methods for differentiating myoblasts often result in randomly arranged myotubes that lack mature sarcomeric structures, but our 3D in vitro culture system provides a mechanical environment similar to in vivo muscle tissue, improving cell differentiation and maturation while enabling high resolution imaging of fixed and live cells.

Materials and

Methods: : Primary mouse myoblasts were isolated from lamin A/C-deficient (Lmna KO) mice and wild type littermates and cultured in a 3D tissue culture device designed to maintain cells suspended in an ECM matrix mixture. The ECM mixture consists of 40% fibrinogen, 40% IMDM, 10% Matrigel, and 10% Geltrex. The ECM mixture is polymerized with 0.5 U/mg thrombin and the cell-ECM solution is seeded into the oval glass-bottom wells of the culture device. After seeding, the cover of the culture device, containing two semi-flexible posts at opposite sides of each oval well, is placed on top and the ECM mixture continues to polymerize and form a strip of tissue suspended between the two posts. The tissues are cultured in myoblast differentiation media for 10-14 days before downstream experiments and analysis are conducted. To observe tissue formation and cell differentiation, tissues were fixed in 4% PFA and stained for desmin, actin, lamin B1 and DAPI, enabling us to monitor cytoskeletal organization and nuclear envelope rupture. To apply electrical pulses to the tissue, electrodes were placed on either end of the oval well and an electrical stimulator was used to apply an alternating pulse wave at either 0.5 Hz or 200 Hz.

Results, Conclusions, and Discussions:: Our 3D in vitro tissue model robustly forms muscle-like tissues from primary myoblasts and is compatible with high-resolution imaging, allowing for observation of nuclear morphology, nuclear envelope rupture, cytoskeletal organization, and mechanoresponsive signaling molecules throughout myoblast differentiation. In addition, this device can apply electrical pacing via electrodes inserted into the wells, allowing application of mechanical stress to the tissue, observing tissue force generation, and improving myotube maturation. During electrical pacing, we observed groups of myotubes consistently and collectively beating within the tissue, suggesting improved maturity compared to 2D in vitro systems.
Most notably, we have used this culture system to develop a novel 3D engineered model of EDMD using primary mouse myoblasts from Lmna KO mice. To investigate the phenotypes present in our model, we stained the engineered tissues for cytoskeletal proteins and lamin B1 to visualize myoblast maturation and nuclear morphology. We observed that Lmna KO engineered tissues form fewer myotubes compared to wild type engineered tissues, consistent with a 2D in vitro culture system. These findings suggests that loss of lamin A/C perturbs myoblast differentiation, potentially resulting from increased nuclear damage which has been observed in vivo. To test the hypothesis that loss of lamin A/C results in increased nuclear damage during myotube formation when cytoskeletal forces on the nucleus increase, we quantified the percentage of nuclei with nuclear envelope rupture marked by the presence of chromatin protrusions from the nuclear envelope. We found that Lmna KO engineered muscle tissue had significantly higher rates of nuclear envelope rupture. These data suggest that loss of lamin A/C may increase myonuclear damage during differentiation in a 3D environment. In addition, these data show that our novel 3D in vitro model of EDMD recapitulates an important phenotype, increased nuclear ruptures. Our model can be used to observe cellular mechanics during myoblast differentiation in an in vivo-like mechanical environment for identifying early disease markers for therapeutic targets. In addition, applying mechanical stress via electrical stimulation can be used to understand how lamin A/C affects myotubes under stress and to study changes in EDMD muscle tissue mechanics.

Acknowledgements and/or References (Optional):: Arne D Hofemeier, Tamara Limon, Till Moritz Muenker, Bernhard Wallmeyer, Alejandro Jurado, Mohammad Ebrahim Afshar, Majid Ebrahimi, Roman Tsukanov, Nazar Oleksiievets, Jörg Enderlein, Penney M Gilbert, Timo Betz (2021) Global and local tension measurements in biomimetic skeletal muscle tissues reveals early mechanical homeostasis eLife 10:e60145