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

Role of Nuclear Lamin A/C in the Regulation of Nav1.5 Channel and Microtubules: Lesson From the Pathogenic Lamin A/C Variant Q517X

In this work, we studied an lmna nonsense mutation encoding for the C-terminally truncated Lamin A/C (LMNA) variant Q517X, which was described in patients affected by a severe arrhythmogenic cardiomyopathy with history of sudden death. We found that LMNA Q517X stably expressed in HL-1 cardiomyocytes abnormally aggregates at the nuclear envelope and within the nucleoplasm. Whole-cell patch clamp experiments showed that LMNA Q517X-expressing cardiomyocytes generated action potentials with reduced amplitude, overshoot, upstroke velocity and diastolic potential compared with LMNA WT-expressing cardiomyocytes. Moreover, the unique features of these cardiomyocytes were 1) hyper-polymerized tubulin network, 2) upregulated acetylated α-tubulin, and 3) cell surface Nav1.5 downregulation. These findings pointed the light on the role of tubulin and Nav1.5 channel in the abnormal electrical properties of LMNA Q517X-expressing cardiomyocytes. When expressed in HEK293 with Nav1.5 and its β1 subunit, LMNA Q517X reduced the peak Na+ current (INa) up to 63% with a shift toward positive potentials in the activation curve of the channel. Of note, both AP properties in cardiomyocytes and Nav1.5 kinetics in HEK293 cells were rescued in LMNA Q517X-expressing cells upon treatment with colchicine, an FDA-approved inhibitor of tubulin assembly. In conclusion, LMNA Q517X expression is associated with hyper-polymerization and hyper-acetylation of tubulin network with concomitant downregulation of Nav1.5 cell expression and activity, thus revealing 1) new mechanisms by which LMNA may regulate channels at the cell surface in cardiomyocytes and 2) new pathomechanisms and therapeutic targets in cardiac laminopathies

Targeting unfolded protein response reverts ER stress and ER Ca2+ homeostasis in cardiomyocytes expressing the pathogenic variant of Lamin A/C R321X

Background: We previously demonstrated that an Italian family affected by a severe dilated cardiomyopathy (DCM) with history of sudden deaths at young age, carried a mutation in the Lmna gene encoding for a truncated variant of the Lamin A/C protein (LMNA), R321X. When expressed in heterologous systems, such variant accumulates into the endoplasmic reticulum (ER), inducing the activation of the PERK-CHOP pathway of the unfolded protein response (UPR), ER dysfunction and increased rate of apoptosis. The aim of this work was to analyze whether targeting the UPR can be used to revert the ER dysfunction associated with LMNA R321X expression in HL-1 cardiac cells. Methods: HL-1 cardiomyocytes stably expressing LMNA R321X were used to assess the ability of 3 different drugs targeting the UPR, salubrinal, guanabenz and empagliflozin to rescue ER stress and dysfunction. In these cells, the state of activation of both the UPR and the pro-apoptotic pathway were analyzed monitoring the expression levels of phospho-PERK, phospho-eIF2α, ATF4, CHOP and PARP-CL. In addition, we measured ER-dependent intracellular Ca2+ dynamics as indicator of proper ER functionality. Results: We found that salubrinal and guanabenz increased the expression levels of phospho-eIF2α and downregulated the apoptosis markers CHOP and PARP-CL in LMNA R321X-cardiomyocytes, maintaining the so-called adaptive UPR. These drugs also restored ER ability to handle Ca2+ in these cardiomyocytes. Interestingly, we found that empagliflozin downregulated the apoptosis markers CHOP and PARP-CL shutting down the UPR itself through the inhibition of PERK phosphorylation in LMNA R321X-cardiomyocytes. Furthermore, upon empagliflozin treatment, ER homeostasis, in terms of ER ability to store and release intracellular Ca2+ was also restored in these cardiomyocytes. Conclusions: We provided evidence that the different drugs, although interfering with different steps of the UPR, were able to counteract pro-apoptotic processes and to preserve the ER homeostasis in R321X LMNA-cardiomyocytes. Of note, two of the tested drugs, guanabenz and empagliflozin, are already used in the clinical practice, thus providing preclinical evidence for ready-to-use therapies in patients affected by the LMNA R321X associated cardiomyocytes