Lamin A/C Mutations Linked to Muscular Disease Result in Mechanically‐induced, Progressive Nuclear Envelope Rupture and DNA Damage in Muscle Fibers

Abstract

Lamin A/C (Lmna) is an inner nuclear membrane protein that confers the stiffness of the nucleus. Mutations in Lmna cause a broad spectrum of human diseases, termed laminopathies, which include various muscular dystrophies and dilated cardiomyopathy. The cause of these tissue‐specific diseases is unclear; however, one potential explanation could be that Lmna mutations alter the stability of nuclei, which is particularly deleterious in mechanically active tissue. To this end, we utilized a novel in vitro model system to differentiate primary myoblasts from Lmna‐KO, Lmna 195K‐ and Lmna H222P –mutant mice into mature, highly contractile myofibers. We found that Lmna mutations alter nuclear stability in primary mouse myoblasts, which persists during differentiation into myofibers, resulting in highly elongated myonuclei and severe nuclear damage. Using both endogenous proteins and live‐cell reporters, we found that Lmna‐mutant myonuclei experience transient nuclear ruptures that progressively increase in number during myofiber maturation. This loss of compartmentalizing was associated with an increase in DNA damage, measured by γH2AX and 53BP1 accumulation, and an overall decrease in myofiber viability. Importantly, these same nuclear defects were also observed in vivo within muscle fibers from our Lmna‐KO, Lmna 195K‐ and Lmna H222P mouse models. This led us to hypothesize that nuclear envelope ruptures are induced by mechanical forces present in differentiated myofibers, resulting in widespread DNA damage and myofiber pathology. Since microtubules are required for nuclear movement, we tested whether microtubules could be involved in inducing myonuclear rupture. We used pharmacological approaches to either destabilize (nocodazole) or stabilize (paclitaxel) the microtubule network in lamin A/C‐mutant myofibers. Interestingly, the destabilization of microtubules increased the amount of nuclear damage and rupture events, while stabilizing microtubules dramatically reduced nuclear damage and rupture events, which also corresponded to an increase and decrease in DNA damage, respectively. Lastly, to investigate whether the DNA damage in Lmna‐mutant myotubes may be underlying their decreased viability, we inhibited the DNA damage repair pathways using ATM or DNA‐PK inhibitors. Inhibiting the activity of DNA‐PK, a kinase required for non‐homologous end joining DNA repair, improved the health of lamin A/C‐mutant myofibers. Taken together, our findings suggest that lamin A/C mutations associated with muscle disease mechanically weaken the nucleus, and result in severe nuclear damage coupled with an increased DNA damage response in muscle fibers that could contribute to the muscle‐specific phenotypes seen in many laminopathies.Support or Funding InformationThis work was supported by the National Institutes of Health (award R01HL082792), donations from the Mills family, and a Fleming postdoctoral fellowship to T.K.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.