Uncovering the Role of Lamin A/C in Nuclear Mechanics and Regulation of Gene Expression in Health and Disease

239 pagesLamin A/C, encoded by the LMNA gene, forms a dense protein meshwork at the nuclear envelope and functions to give the nucleus structural support, organize chromatin, and aid diverse cellular signaling pathways. Lamin A/C are particularly critical for maintaining the mechanical properties of the nucleus, and in their role as a mechanosensor of cellular forces, thereby enabling the nucleus to physically and biochemically adapt to its mechanical environment. As such, mutations in LMNA cause a host of human diseases, termed ‘laminopathies,’ that include Dilated Cardiomyopathy (LMNA-DCM) and several forms of muscular dystrophy, among others. However, an incomplete understanding of the cellular disease mechanisms driving DCM and other laminopathies has resulted in a lack of therapeutics sufficient to ameliorate disease pathogenesis. The primary goal of my thesis has been to understand changes to the mechanical conformation of nuclei and to gene expression that may be driving nuclear and cellular dysfunction in LMNA-DCM. Using an induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) derived from LMNA-DCM patients with several different gene mutations, I found that the mislocalization of Lamin A/C and Lamin B1 from the nuclear envelope in LMNA-mutant iPSC-CMs corresponds to an increase in nuclear damage and fragility, likely due to the inability of the nucleus to withstand mechanical forces. Additionally, as Lamin A/C are heavily involved in control of gene expression and downstream cellular signaling pathways, through RNA-sequencing of cardiac tissue from several Lmna-DCM mouse models and iPSC-CMs carrying the LMNA G449V mutation, I found that metabolism, extracellular matrix, cardiac, and immune response genes and associated signaling pathways are misregulated in both mouse and human models of LMNA Dilated Cardiomyopathy, and misregulated metabolism and extracellular matrix are involved in the disease onset and progression of Lmna N195K mice. A secondary goal of my thesis work was to understand how Lamin A/C mediates the transduction of forces to the nucleus. I found that the Lamin A/C Ig-fold undergoes conformational changes in response to cell seeding density, which allows for the differential binding of antibodies targeting the Ig-fold. As the Ig-fold is home to the binding sites of numerous nuclear proteins involved in mechanotransduction of forces and gene expression and is a hotspot for LMNA mutations that affect nuclear mechanotransduction, clarifying the role and context of conformational changes in the context of healthy mechanotransduction is critical for progress towards a complete understanding of disrupted nuclear organization and mechanotransduction in laminopathies. Together, these studies represent key ways in which Lamin A/C impacts nuclear mechanics and mechanotransduction in health and disease, and exhibit several novel mechanisms through which LMNA mutations drive changes in gene expression to cause cardiac dysfunction, progress which is necessary for the development of novel therapeutics for laminopathies which sufficiently target the underlying cellular disease pathology