LINC complex disruption can improve or even prevent dilated cardiomyopathy caused by lamin mutations

Introduction:: Mutations in the LMNA gene, encoding the nuclear envelope (NE) proteins lamins A/C, are responsible for a plethora of human diseases (laminopathies), often specifically affecting striated muscle. The most prevalent laminopathy is LMNA-related dilated cardiomyopathy (LMNA-DCM), with several other laminopathies also exhibiting cardiac defects. The molecular mechanisms leading to the striated muscle specific pathogenesis remain largely unresolved, leaving patients with limited and often ineffective treatment options. Cardiac muscle biopsies from patients and mouse models of LMNA-DCM provide anecdotal evidence of nuclear defects. In addition, we had previously shown that LMNA mutations can result in mechanically more fragile nuclei, and that disruption of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex using a dominant negative nesprin KASH domain (KASH) can reduce NE rupture and improve skeletal muscle function and viability in vitro (Earle et al. Nature Materials 2020). It remains unclear, however, whether nuclear damage causally contributes to disease progression in LMNA-DCM, and whether reducing cytoskeletal forces on the more fragile nuclei can prevent NE rupture and reduce or prevent cardiac defects in lamin A/C deficient or mutant hearts.


Materials and Methods:: To test the hypothesis that LMNA mutations reduce nuclear stability and cause transient NE rupture in cardiac (and skeletal) muscle cells, causing DNA damage and promoting pathogenesis, we generated a novel mouse model that expresses a transgenic cGAS-tdTomato (cGAS) reporter construct that accumulates at sites of NE rupture and persists even after the NE has been repaired. The cGAS mouse model was then crossed into two established striated muscle laminopathy mouse models, a complete deletion (Lmna KO) and a knock-in model (Lmna N195K). Both mouse models develop muscular dystrophy and DCM with varying severities. To assess mutation- and tissue-specific NE rupture events, we applied a novel whole tissue imaging protocol combining tissue clearing and lightsheet imaging. In a complementary approach to study the role of lamins and mechanical load in cardiac disease pathogenesis, we generated a novel mouse model with tamoxifen inducible, cardiac specific deletion of lamin A/C, which was then crossed with the cGAS mouse model. To induce cardiac-specific LINC complex disruption in the diverse mouse models, we crossed the above models with mice with a Cre-inducible KASH transgene, along with a tamoxifen inducible, cardiac specific Cre recombinase (α-myosin heavy chain mER-Cre-mER, abbreviated as Cre). We then assessed cardiac histology, function, and overall survival in the various Lmna mutant and wild-type mice in the absence or presence of KASH expression.


Results, Conclusions, and Discussions:: Whereas NE rupture was virtually non-existent in wild-type mice, Lmna KO and Lmna N195K mice had significant increased incidents of NE rupture events, indicated by the presence of cGAS-tdTomato foci (Figure 1A) in skeletal and cardiac muscle. In contrast, NE rupture was absent in the brain and liver of these animals (Figure 1B-C), suggesting that NE rupture is specific to tissues subjected to high mechanical stress. This provides a potential explanation for the highly muscle specific defects in laminopathies. Supporting a causative role of the increased NE rupture in driving cardiac dysfunction, cardiac specific LINC complex disruption in the Lmna N195K mice, which reduces cytoskeletal forces on the muscle nuclei, resulted in significantly increased survival (Figure 1D). Since pre-existing muscle damage, plus effects of the Lmna N195K mutation on other tissues could explain the only partial rescue, we also assessed LINC complex disruption in a cardiac-specific inducible lamin A/C deletion model (αMHC-Cre;Lmna^fl/fl). Cardiac specific deletion of lamin A/C in adult mice resulted in the rapid development of severe dilated cardiomyopathy and sudden death. Echocardiographic and histological analysis revealed reduced ejection fraction, left ventricle thinning and dilatation, interstitial fibrosis, increased incidence of NE rupture events, activation of DNA damage response, and cardiomyocyte vacuolation and loss in the lamin A/C depleted hearts. Intriguingly, reducing cytoskeletal forces on muscle nuclei by LINC complex disruption using a dominant negative nesprin construct (KASH) completely prevented the fatal disease development in the inducible, cardiac specific Lmna deletion model (Figure 1E).

In conclusion, our findings strongly support the idea that mechanical stress from cytoskeletal forces leads to NE rupture in the more fragile lamin A/C deficient and mutant nuclei and promotes pathogenesis in mechanically stressed tissues. Reducing the mechanical stress by LINC complex disruption ameliorates or even completely prevents LMNA-DCM pathogenesis. In addition to reducing NE rupture, LINC complex disruption may also have other beneficial effects on LMNA mutant nuclei, through yet to be determined mechanisms. Regardless of the specific mechanism, our data suggest that LINC complex disruption may serve as a promising, novel therapeutic approach for LMNA-DCM and other striated muscle laminopathies.


Acknowledgements (Optional): : This work was supported funding from the National Institutes of Health (R01 HL082792), the National Science Foundation (MCB-1715606), and generous gifts from the Mills family.


References (Optional): : Earle AJ, Kirby TJ, Fedorchak GR, Isermann P, Patel J, Iruvanti S, Moore SA, Bonne G, Wallrath LL, Lammerding J. Mutant lamins cause nuclear envelope rupture and DNA damage in skeletal muscle cells. Nature Materials. 2020. 19(4): 464-473. doi: 10.1038/s41563-019-0563-5