RESUMO
Surgical intervention is necessary following nerve trauma. Tubular prostheses can guide growing axons and inserting substances within these prostheses can be positive for the regeneration, making it an alternative for the current standard tools for nerve repair. Our aim was to investigate the effects of fibrin glue BthTL when combined with a synthetic TNF mimetic-action peptide on nerve regeneration. Male Wistar rats suffered left sciatic nerve transection. For repairing, we used empty silicon tubes (n = 10), tubes filled with fibrin glue BthTL (Tube + Glue group, n = 10) or tubes filled with fibrin glue BThTL mixed with TNF mimetic peptide (Tube + Glue + Pep group, n = 10). Animals were euthanized after 45 days. We collected nerves to perform immunostaining (neurofilament, GAP43, S100-ß, NGFRp75 and Iba-1), light and transmission electron microscopy (for counting myelinated, unmyelinated and degenerated fibers; and for the evaluation of morphometric aspects of regenerated fibers) and collagen staining. All procedures were approved by local ethics committee (protocol 063/17). Tube + Glue + Pep group showed intense inflammatory infiltrate, higher Iba-1 expression, increased immunostaining for NGFRp75 receptor (which characterizes Schwann cell regenerative phenotype), higher myelin thickness and fiber diameter and more type III collagen deposition. Tube + Glue group showed intermediate results between empty tube and Tube + Glue + Pep groups for anti-NGFRp75 immunostaining, inflammation and collagen; on fiber counts, this group showed more degenerate fibers and fewer unmyelinated axons than others. Empty tube group showed superiority only in GAP43 immunostaining. A combination of BthTL glue and TNF mimetic peptide induced greater axonal regrowth and remyelination.
Assuntos
Adesivo Tecidual de Fibrina , Regeneração Nervosa/efeitos dos fármacos , Peptidomiméticos/administração & dosagem , Peptidomiméticos/farmacologia , Nervos Periféricos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/farmacologia , Animais , Axônios/efeitos dos fármacos , Colágeno/metabolismo , Imuno-Histoquímica , Masculino , Bainha de Mielina/efeitos dos fármacos , Fibras Nervosas Mielinizadas/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Peptidomiméticos/química , Ratos , Ratos Wistar , Células de Schwann/efeitos dos fármacos , Células de Schwann/ultraestrutura , Nervo Isquiático/lesões , Fator de Necrose Tumoral alfa/químicaRESUMO
BACKGROUND: L-type calcium channels (LTCCs) play important roles in regulating cardiomyocyte physiology, which is governed by appropriate LTCC trafficking to and density at the cell surface. Factors influencing the expression, half-life, subcellular trafficking, and gating of LTCCs are therefore critically involved in conditions of cardiac physiology and disease. METHODS: Yeast 2-hybrid screenings, biochemical and molecular evaluations, protein interaction assays, fluorescence microscopy, structural molecular modeling, and functional studies were used to investigate the molecular mechanisms through which the LTCC Cavß2 chaperone regulates channel density at the plasma membrane. RESULTS: On the basis of our previous results, we found a direct linear correlation between the total amount of the LTCC pore-forming Cavα1.2 and the Akt-dependent phosphorylation status of Cavß2 both in a mouse model of diabetic cardiac disease and in 6 diabetic and 7 nondiabetic cardiomyopathy patients with aortic stenosis undergoing aortic valve replacement. Mechanistically, we demonstrate that a conformational change in Cavß2 triggered by Akt phosphorylation increases LTCC density at the cardiac plasma membrane, and thus the inward calcium current, through a complex pathway involving reduction of Cavα1.2 retrograde trafficking and protein degradation through the prevention of dynamin-mediated LTCC endocytosis; promotion of Cavα1.2 anterograde trafficking by blocking Kir/Gem-dependent sequestration of Cavß2, thus facilitating the chaperoning of Cavα1.2; and promotion of Cavα1.2 transcription by the prevention of Kir/Gem-mediated shuttling of Cavß2 to the nucleus, where it limits the transcription of Cavα1.2 through recruitment of the heterochromatin protein 1γ epigenetic repressor to the Cacna1c promoter. On the basis of this mechanism, we developed a novel mimetic peptide that, through targeting of Cavß2, corrects LTCC life-cycle alterations, facilitating the proper function of cardiac cells. Delivery of mimetic peptide into a mouse model of diabetic cardiac disease associated with LTCC abnormalities restored impaired calcium balance and recovered cardiac function. CONCLUSIONS: We have uncovered novel mechanisms modulating LTCC trafficking and life cycle and provide proof of concept for the use of Cavß2 mimetic peptide as a novel therapeutic tool for the improvement of cardiac conditions correlated with alterations in LTCC levels and function.