Functional characterization of Lamin A/C mutants involved in hereditary-familial cardiomyopathies for the development of personalized diagnostic and therapeutic approaches

Nuclear lamin A and C are important components of the multifunctional scaffolds that mechanically supports the inner nuclear membrane, providing nuclear and cytosolic rigidity. However, lamins escape to broader spectrum of functions beyond mere mechanics, also being associated with other physiological processes, such as modulating gene expression and intracellular signaling pathways. To further highlight its key role in cell physiology, mutations in the lamin A/C gene (Lmna) have been associated with a variety of pathological phenotypes with the skeletal muscles and the heart being the most affected systems. When affected, the heart can develop a wide range of phenotypes, from dilated cardiomyopathy with conduction defects (DCM-CD) to arrhythmogenic right ventricular cardiomyopathy. The variety of cardiac phenotypes is likely the consequence of the significant number of different Lmna mutations identified so far (1). Given the complexity of obtaining reliable genotype-phenotype correlations in Lmna-related diseases, it is crucial to functionally characterize each mutation to better understand its specific effects and tailor treatment strategies accordingly (personalized medicine). In this study, we analysed three different Lmna mutations to shed light on their specific pathogenetic mechanism at the cellular level. These mutations have been associated with different phenotypic outcome, all characterized by a consistent recurrence of dilated cardiomyopathy (DCM) with a poor prognosis. The nonsense mutation LMNA Q517X abnormally aggregates at the nuclear envelope and within the nucleoplasm of HL-1 cardiomyocytes. In addition, LMNA Q517X-expressing cardiomyocytes are characterized by hyper-polymerized tubulin network, upregulation of acetylated α-tubulin, downregulation of Nav1.5 channels on the cell surface and significant changes in action potential parameters, indicating abnormal electrical properties. Further examination in HEK293 cells expressing LMNA Q517X with Nav1.5 shows a significant reduction in peak Na+ current (INa) and altered channel kinetics. Treatment with colchicine, an FDA-approved tubulin assembly inhibitor, rescues cellular properties and channel kinetics in LMNA Q517X-expressing cardiomyocytes. Then, we investigated the molecular basis of LMNA R321X-associated DCM and explored potential pharmacological interventions that target the unfolded protein response (UPR). We demonstrated the activation of the PERK-CHOP pathway of the UPR and subsequent endoplasmic reticulum (ER) dysfunction and apoptosis in HL-1 cardiomyocytes that stably express LMNA R321X. We assessed the effectiveness of three UPR-targeting drugs-salubrinal, guanabenz, and empagliflozin-in alleviating ER stress. Salubrinal and guanabenz function maintaining adaptive UPR state, reducing ER stress and pro-apoptotic markers, and restoring ER calcium handling. Empagliflozin suppresses apoptosis markers and inhibits the UPR by blocking PERK phosphorylation in LMNA R321X cardiomyocytes. Additionally, empagliflozin treatment restores endoplasmic reticulum (ER) homeostasis, allowing it to properly store and release intracellular calcium ions (Ca2+). The study shows that various drugs targeting different stages of the UPR can effectively reduce pro-apoptotic pathways and maintain ER homeostasis in R321X LMNA-cardiomyocytes. It is noteworthy that guanabenz and empagliflozin, which are already in clinical use, offer promising therapeutic options for LMNA R321X-associated cardiomyopathies. Finally, we investigated the molecular and cellular mechanisms underlying Emery-Dreifuss muscular dystrophy (EDMD), which is characterized by slowly progressive muscle weakness and wasting and DCM. We used a knock-in LmnaH222P/H222P mouse model carrying the LMNA p.H222P mutation, which recapitulates all the features of EDMD and also key features of cardiac laminopathies. We observed altered tyrosinated α-tubulin network in muscle fibers of these mice compared to wild-type, indicating disrupted microtubule organization. Here, we aim to understand how abnormal microtubule organization contributes to nuclear elongation through impaired interaction with microtubule-associated proteins (MAPs). Our investigation revealed distinct localization patterns of CLIP-170, a MAP, in the muscle fibers of LmnaH222P/H222P mice compared to wild-type. Pharmacological modulation of CLIP-170 activity with a neurosteroid restores CLIP-170 localization and nuclear morphology in the muscle fibers of mice carrying the LMNA p.H222P mutation, highlighting its therapeutic potential in improving EDMD-associated phenotypes. Collectively, this thesis provides comprehensive insights into the pathogenic mechanisms of Lmna-associated cardiomyopathies and identifies potential therapeutic strategies, ranging from tubulin-targeting agents to UPR modulators and CLIP-170 regulators, offering hope for the development of effective treatments for these devastating diseases.