Mechanistic Insights of the LEMD2 p.L13R Mutation and Its Role in Cardiomyopathy

Abstract

BACKGROUND: Nuclear envelope proteins play an important role in the pathogenesis of hereditary cardiomyopathies. Recently, a new form of arrhythmic cardiomyopathy caused by a homozygous mutation (p.L13R) in the inner nuclear membrane protein LEMD2 was discovered. The aim was to unravel the molecular mechanisms of mutant LEMD2 in the pathogenesis of cardiomyopathy. METHODS: We generated a Lemd2 p.L13R knock-in mouse and a corresponding cell model via CRISPR/Cas9 technology and investigated the cardiac phenotype as well as cellular and subcellular mechanisms of nuclear membrane rupture and repair. RESULTS: Knock-in mice developed a cardiomyopathy with predominantly endocardial fibrosis, left ventricular dilatation, and systolic dysfunction. Electrocardiograms displayed pronounced ventricular arrhythmias and conduction disease. A key finding of knock-in cardiomyocytes on ultrastructural level was a significant increase in nuclear membrane invaginations and decreased nuclear circularity. Furthermore, increased DNA damage and premature senescence were detected as the underlying cause of fibrotic and inflammatory remodeling. As the p.L13R mutation is located in the Lap2/Emerin/Man1 (LEM)-domain, we observed a disrupted interaction between mutant LEMD2 and BAF (barrier-to-autointegration factor), which is required to initiate the nuclear envelope rupture repair process. To mimic increased mechanical stress with subsequent nuclear envelope ruptures, we investigated mutant HeLa-cells upon electrical stimulation and increased stiffness. Here, we demonstrated impaired nuclear envelope rupture repair capacity, subsequent cytoplasmic leakage of the DNA repair factor KU80 along with increased DNA damage, and recruitment of the cGAS (cyclic GMP–AMP synthase) to the nuclear membrane and micronuclei. CONCLUSIONS: We show for the first time that the Lemd2 p.L13R mutation in mice recapitulates human dilated cardiomyopathy with fibrosis and severe ventricular arrhythmias. Impaired nuclear envelope rupture repair capacity resulted in increased DNA damage and activation of the cGAS/STING/IFN pathway, promoting premature senescence. Hence, LEMD2 is a new player in the disease group of laminopathies.

Pervasive nuclear envelope ruptures precede ECM signaling and disease onset without activating cGAS-STING in Lamin-cardiomyopathy mice

“…Reduction of nuclear lamins can cause ruptures of the nuclear envelope (NE ruptures) [28][29][30][31][33][34][35] . Indeed, NE ruptures have been observed in patients 17,18,32,36,37 , animal models 30,31,[38][39][40][41][42] , and cell culture models 30,35 of LMNA-related DCM and laminopathies. However, the extent of NE ruptures in LMNArelated DCM and the contribution of this event to the pathogenesis of DCM remain undefined.…”

“…A prevailing hypothesis for LMNA-related DCM pathogenesis implicates the cGAS-STING pathway as a link between NE rupture, inflammatory signaling, and DCM pathogenesis 39,40,[43][44][45] . cGAS-STING is a cytosolic DNA-sensing innate immune mechanism, in which cGAS binds to cytosolic DNA and activates cell-autonomous STING-mediated interferon transcription 46,47 .…”

A Novel Role for CSA in the Regulation of Nuclear Envelope Integrity: Uncovering a Non-Canonical Function

“…Mutations in LEMD2 have also been linked to cardiomyopathy in humans (56). For instance, patients carrying homozygous mutation (c.38T>G, p.L13R) in the LEMD2 gene develop ventricular arrhythmia and fibrosis (57). It is noteworthy to mention that the L13R mutation causes a significant reduction in LEMD2 expression in cardiomyocytes and causes NE defects including nuclear membrane invaginations and decreased nuclear circularity.…”

The Role of the LEMD2 p.L13R Mutation in Dilated Cardiomyopathy

“…The C-terminal part of LEMD2 has an important role in the repair of nuclear envelope ruptures (NERs) by recruiting the ESCRT (endosomal sorting complex required for transport)-III. 11 The aim of the present study 13 was to further elucidate the molecular mechanisms of the cardiomyopathy caused by the homozygous LEMD2 p.L13R mutation. The authors generated a LEMD2 p.L13R knock-in (KI) mouse to replicate the human condition.…”

“…The aim of the present study 13 was to further elucidate the molecular mechanisms of the cardiomyopathy caused by the homozygous LEMD2 p.L13R mutation. The authors generated a LEMD2 p.L13R knock-in (KI) mouse to replicate the human condition.…”