LINC complex disruption improves cardiac function and overall survival in striated muscle disease caused by lamin mutations

Introduction: Mutations in the LMNA gene, encoding the nuclear envelope 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 (DN 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. To address this question, we generated several novel mouse models of striated muscle laminopathies with inducible expression of a DN KASH construct and then characterized the effect of DN KASH expression on cardiac and skeletal muscle function and overall survival in these mice.

Materials and

Methods: To test the hypothesis that reducing cytoskeletal forces on lamin mutant muscle cell nuclei improves striated muscle health and overall survival in laminopathies, we assessed the effect of LINC complex disruption through expression of a DN KASH construct in three different mouse models: (1) mice with systemic, constitutive deletion of the Lmna gene (Lmna KO); (2) mice generated in our laboratory with cardiac specific, tamoxifen (TMX) inducible Lmna deletion (cLmna KO); and (3) mice expressing an Lmna mutation responsible for striated muscle disease (Lmna N195K). Lmna KO mice develop severe striated muscle disease and normally die at 4-8 weeks of age; cLmna KO mice typically die within 3-4 weeks of tamoxifen injection; Lmna N195K mice develop striated muscle disease and normally die around 12 weeks of age. We crossed these mouse models with animals harboring a Cre-inducible DN KASH construct and a tamoxifen inducible Cre construct driven by either a cardiac specific promoter (alpha-myosin heavy chain, aMHC-MCM) or a skeletal muscle specific promoter (alpha-skeletal actin, HSA-MCM). Cre-expression was induced by repeated injection of tamoxifen either shortly after birth (for the Lmna KO mice), at 3-weeks of age (Lmna N195K), or at 10 weeks of age (cLmna KO). We then assessed cardiac and skeletal muscle histology, function, and overall survival in the various Lmna mutant and wild-type mice in the absence or presence of KASH expression. In addition, we assessed NE rupture in striated muscle tissue based on histological analysis and expression of a transgenic fluorescent NE rupture reporter (cGAS-tdTomato).

Results, Conclusions, and Discussions: Whereas NE rupture was virtually non-existent in wild-type mice, the Lmna KO, cLmna KO, and Lmna N195K mice had significant increased incidents of NE rupture, indicated by the presence of cGAS-tdTomato foci and chromatin protrusions in skeletal and cardiac muscle (Fig.1A-C). DN KASH expression significantly reduced the fraction of damaged skeletal and cardiac muscle nuclei. For all models, we confirmed LINC complex disruption by displacement of endogenous nesprins from the NE (Fig.1E). Supporting a causative role of the increased NE rupture in driving cardiac dysfunction, LINC complex disruption, which reduces cytoskeletal forces on the muscle nuclei, resulted in significantly increased survival in the Lmna KO and Lmna N195K mice (Fig.2A-B). Since pre-existing muscle damage, plus effects of the systemic Lmna N195K mutation or Lmna deletion on other tissues could explain the only partial rescue effect, we also assessed the effect of LINC complex disruption in the cLmna KO model. Without LINC complex disruption, cardiac specific Lmna deletion in adult mice (Fig.1D) resulted in rapid development of severe dilated cardiomyopathy and sudden death (Fig.2C-D). Echocardiographic and histological analysis revealed reduced ejection fraction, left ventricle thinning and dilatation, interstitial fibrosis, increased incidence of NE rupture events, and DNA damage in the lamin A/C depleted hearts. LINC complex disruption using the DN KASH construct dramatically improved cardiac function and extended survival in the cLmna KO mice by more than 12 months, showing a dramatic rescue effect (Fig.2C-D). Collectively, 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 almost completely eliminated LMNA-DCM pathogenesis when administered concurrently with Lmna deletion, but had more limited, although still striking rescue effects in the other models. 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. 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.