Dilated cardiomyopathy (DCM) associates left ventricular dilatation and systolic dysfunction. It is a major cause of heart failure and cardiac transplantation. LMNA is the most frequent gene involved in DCM with conduction/rhythm defects. It encodes nuclear envelope proteins, the lamin A/C. The mechanisms linking LMNA mutations to DCM remain to be elucidated. We generated a mouse model deleted of lysine 32 (delK32). Heterozygous (Het) mice developed DCM leading to death between 35 and 70 weeks of age. The aim of this study was to evaluate the expression of the mutant in mouse heart during the course of the disease and its impact on contractile properties of fibrin-based rat engineered heart tissues (EHT).Methods
Lamin A/C protein levels were measured by Western blot. EHT were transduced with adeno-associated virus encoding Flag-tagged wild-type (Wt) or delK32 human pre-lamin A. Contractile properties were measured using a video set-up for 17 days after transduction. Structure of EHT and nuclei was evaluated by immunofluorescence.Results
Cardiac lamin A/C protein level was 50% lower in Het than in Wt before the onset of DCM, but did not differ from Wt after. We hypothesised that mutant lamin is degraded by the ubiquitin-proteasome system (UPS), leading to UPS impairment and accumulation of poison delK32 lamin A/C with age in Het mice. Wt-, and to a lower extent delK32-, lamin A-transduced EHT showed impaired contractile function compared to untransduced EHTs (-80% and -62% of force, respectively). In EHT, Wt lamin A had rim nuclear localization, whereas delK32 lamin A showed low diffuse expression within the nucleoplasm. Treatment of EHT with the proteasome inhibitor epoxomicin had no major impact on Wt lamin A localization, whereas it increased the level of delK32 lamin A, which formed aggregates into the nuclei. This suggests degradation of delK32 lamin A via the UPS, supporting our hypothesis of degradation of endogenous mutant lamin in vivo.Conclusion
Overexpression of lamin A/C is known to be detrimental for cell function. The smaller effect of delK32 than Wt on EHT contractile properties might be explained by lower amount of delK32 lamin A protein due to degradation via the UPS. In young mice, endogenous mutant lamin is probably degraded by UPS, while age-related impairment of UPS in older mice might lead to accumulation of mutant lamin with a poison action on contractile function. Preliminary results of mutant lamin quantification in Het hearts seem to confirm this hypothesis.