Lmna deficiency promotes EPHX2 nuclear translocation to ameliorate cardiac dysfunction in mice

Abstract

BackgroundCardiovascular diseases are often associated with altered protein subcellular localization. As a major cause of inherited cardiomyopathy, LMNA deficiency could trigger nuclear envelope rupture and broadly impair the localization of nuclear and cytoplasmic proteins. Systemic approaches to identify, dissect and manipulate the localization of endogenous proteins are important for mechanistic and therapeutic investigation. MethodProximity proteomics of the nuclear lamina was performed specifically in cardiomyocytes in Lmna-deficient murine models. AAV-mediated Cas9-based gene silencing and subcellular gene upregulation were conducted via the nuclear localization signal (NLS) and nuclear export signal (NES). Cas9-based somatic mutagenesis was supplemented with the single-strand DNA templates of AAV to achieve robust homology-directed repair (HDR) and targeted NLS knock-in, which translocated cytoplasmic proteins into nuclei. ResultIn vivo proximity proteomics detected increased epoxide hydrolase 2 (EPHX2) in cardiomyocyte nuclei in mice carrying germline or cardiac-specific Lmna truncating variants. This phenotype was associated with ruptured nuclear envelope. Cas9-mediated Ephx2 knockout in cardiomyocytes ameliorated cardiac dysfunction in Lmna-deficient mice. Strikingly, overexpression of NLS-EPHX2, but not NES-EPHX2, also mitigated cardiac dysfunction. The cardiac protective EPHX2 substrates, epoxyeicosatrienoic acids (EETs), did not alter upon NLS-EPHX2 overexpression. By contrast, the Lmna-related DNA damage marker {gamma}-H2AX was reduced. The EPHX-D333A mutant lacking hydrolase activity recapitulated the effects of wildtype EPHX2 in nuclei. AAV-Cas9-based HDR achieved efficient NLS knock-in and EPHX2 nuclear translocation in more than 60% cardiomyocytes, which improved cardiac function. ConclusionLmna deficiency leads to the nuclear translocation of EPHX2, which ameliorated cardiac dysfunction in a hydrolase-independent manner. AAV-HDR-mediated somatic gene editing provides an efficient approach to manipulate the subcellular localization of endogenous proteins in cardiomyocytes in vivo. What is Known?O_LICardiovascular diseases are regulated by the changes in protein subcellular localization. In particular, LMNA-related cardiomyopathy is associated with nuclear rupture and impaired separation between nuclear and cytoplasmic proteins. C_LIO_LIEpoxide hydrolase 2 (EPHX2) is a cytoplasmic hydrolase that catalyzes the hydrolysis of cardioprotective epoxyeicosatrienoic acids (EETs) and aggravates an array of heart diseases including myocardial infarction and heart failure. C_LIO_LICRISPR/Cas9-mediated homology-directed repair (HDR) exhibits uniquely high gene editing efficiency in cardiomyocytes with an AAV-based DNA donor, which is suitable for somatic genetic knock-in of small DNA fragments in vivo. C_LI What New Information Does This Article Contribute?O_LILamin-targeted proximity proteomics specifically in cardiomyocytes uncovers novel proteins undergoing subcellular localization changes upon Lmna deficiency. C_LIO_LILmna deficiency leads to EPHX2 nuclear translocation that ameliorates cardiac dysfunction by both mechanisms of cytoplasmic reduction and nuclear induction. C_LIO_LIAAV-HDR-mediated knock-in provides a robust platform to manipulate subcellular localization of endogenous proteins specifically in cardiomyocytes in vivo. C_LI Cardiovascular diseases are often associated with altered protein subcellular localization, but systemic approaches to identify, study and manipulate subcellular localization remain incomplete. This study established an in vivo proximity proteomics approach to identify novel proteins undergoing localization changes relative to the nuclear lamina. In murine models of LMNA-related cardiomyopathy, this approach uncovered EPHX2, a classic cytoplasmic hydrolase that aggravates cardiovascular diseases, as a new protein that translocated into cell nucleus and exerted a cardiac protective effect. CRISPR/Cas9-based cardiomyocyte gene editing with an AAV-based DNA donor efficiently achieved NLS knock-in into the Ephx2 gene, promoted EPHX2 protein nuclear translocation and mitigated cardiac dysfunction with Lmna deficiency. These findings indicated a novel avenue to identify and manipulate the subcellular localization changes of endogenous proteins for basic and translational cardiology.