ALTERED NUCLEAR AND CYTOSKELETAL MECHANICS IN CARDIAC MYOCYTES WITH D192G NUCLEAR LAMIN MUTATION



Carlin S. Long, M.D., University of Colorado, Denver, CO, USA

Atomic force microscopy whole-cell loading/unloading curves were used to study the mechanical behavior of cardiomyocytes carrying the LMNA D192G mutation which is known to cause a severe form of dilated cardiomyopathy. Here, combining atomic force microscopy (AFM) with molecular and cellular biology methodologies, we studied both nuclear and whole-cell biomechanical behavior in Neonatal rat ventricular myocytes (NRVMs) expressing the LMNA D192G mutation and compared this with both control cells and those expressing wild-type LMNA. LMNA protein expression was confirmed up to day 6. Live-cell AFM force-deformation curves from days 1 through 6 showed that LMNA D192G nuclei displayed increased stiffness compared to controls with a peak at 72 hours (p < 0.05), with a 3 time increase in nuclear Young modulus. Furthermore, mutant NRVMs showed an unexpected reduction in the adhesion area between AFM probe and cell membrane compared to control and wild-type. Finally, D192G NRVMs displayed altered cytoskeletal deformation measured as force decays with time (relaxation force test) compared to wild-type and control NRVMs, suggesting loss of cytoskeleton elasticity. The altered mechanical behavior of LMNA D192G NRVMs was rescued by wild-type LMNA expression in mutant cells. Our results suggest that the LMNA D192G mutation has a profound effect on the whole-cell biomechanics in cardiomyocytes, extending beyond the increased nuclear stiffness, indicating cytoskeletal structural modifications and reduced cell membrane adhesion, changes that can be rescued by wild-type LMNA. These findings extend our understanding of the pathophysiology of this, and perhaps other, gene-specific causes of cardiomyopathy and provide a cell-based assay for their analysis and potential novel therapies.