AFM single-cell force spectroscopy links altered nuclear and cytoskeletal mechanics to defective cell adhesion in cardiac myocytes with a nuclear lamin mutation

Previous investigations suggested that lamin A/C gene (LMNA) mutations, which cause a variety of human diseases including muscular dystrophies and cardiomyopathies, alter the nuclear mechanical properties. We hypothesized that biomechanical changes may extend beyond the nucleus. Combining atomic force microscopy (AFM), molecular and cellular biology, we studied the biomechanical properties of cardiomyocytes expressing the cardiomyopathy LMNA D192G mutation, and attempted rescue through the subsequent introduction of wild-type LMNA. Neonatal rat ventricular myocytes (NRVMs) were infected with adenoviral vectors carrying either human LMNA wild-type or D192G gene. LMNA protein expression was confirmed up to day 6 by western blot. Live-cell AFM force-deformation curves from day 1 through day 6 showed that the nuclei of NRVMs expressing LMNA D192G displayed increased stiffness compared to both uninfected and wild-type expressing cells, with a peak at 48 hours (3-fold increase in nuclear Young modulus, p<0.0001). Furthermore, mutant NRVMs showed a significant reduction in the adhesion area between AFM probe and cell membrane, impaired cytoskeletal deformation measured by relaxation force test, associated with alteration of the cytoskeletal actin network by confocal microscopy. The altered actin network and mechanical properties of LMNA D192G NRVMs were rescued by the subsequent expression of wild-type LMNA. In conclusion, mutant LMNA deleterious effects appear to extend beyond the increased nuclear stiffness, to include altered cytoskeletal mechanics and defective cell membrane adhesion work, observations that are likely to underpin the changes in cardiac function that characterize this severe cardiomyopathy. Finally, expression of wild-type LMNA restores the mechanical properties of mutant NRVMs.