RESUMO
Lamins A and C are nuclear intermediate filament proteins that form a proteinaceous meshwork called lamina beneath the inner nuclear membrane. Mutations in the LMNA gene encoding lamins A and C cause a heterogenous group of inherited degenerative diseases known as laminopathies. Previous studies have revealed altered cell signaling pathways in lamin-mutant patient cells, but little is known about the fate of mutant lamins A and C within the cells. Here, we analyzed the turnover of lamins A and C in cells derived from a dilated cardiomyopathy patient with a heterozygous p.S143P mutation in LMNA. We found that transcriptional activation and mRNA levels of LMNA are increased in the primary patient fibroblasts, but the protein levels of lamins A and C remain equal in control and patient cells because of a meticulous interplay between autophagy and the ubiquitin-proteasome system (UPS). Both endogenous and ectopic expression of p.S143P lamins A and C cause significantly reduced activity of UPS and an accumulation of K48-ubiquitin chains in the nucleus. Furthermore, K48-ubiquitinated lamins A and C are degraded by compensatory enhanced autophagy, as shown by increased autophagosome formation and binding of lamins A and C to microtubule-associated protein 1A/1B-light chain 3. Finally, chaperone 4-PBA augmented protein degradation by restoring UPS activity as well as autophagy in the patient cells. In summary, our results suggest that the p.S143P-mutant lamins A and C have overloading and deleterious effects on protein degradation machinery and pharmacological interventions with compounds enhancing protein degradation may be beneficial for cell homeostasis.
RESUMO
Therapeutic angiogenesis induced by gene therapy is a promising approach to treat patients suffering from severe coronary artery disease. In small experimental animals, adeno-associated viruses (AAVs) have shown good transduction efficacy and long-term transgene expression in heart muscle and other tissues. However, it has been difficult to achieve cardiac-specific angiogenic effects with AAV vectors. We tested the hypothesis whether AAV2 gene transfer (1 × 1013 vg) of vascular endothelial growth factor B (VEGF-B186) together with immunosuppressive corticosteroid treatment can induce long-term cardiac-specific therapeutic effects in the porcine ischemic heart. Gene transfers were delivered percutaneously using direct intramyocardial injections, improving targeting and avoiding direct contact with blood, thus reducing the likelihood of immediate immune reactions. After 1- and 6-month time points, the capillary area was analyzed, myocardial perfusion reserve (MPR) was measured with radiowater positron emission tomography ([15O]H2O-PET), and fluorodeoxyglucose ([18F]FDG) uptake was used to evaluate myocardial viability. Clinical chemistry and immune responses were analyzed using standard methods. After 1- and 6-month follow-up, AAV2-VEGF-B186 gene transfer failed to induce angiogenesis and improve myocardial perfusion and viability. Here, we show that inflammatory responses attenuated the therapeutic effect of AAV2 gene transfer by significantly reducing successful transduction and long-term gene expression despite the efforts to reduce the likelihood of immune reactions and the use of targeted local gene transfer methods.
