RESUMEN
Restoring expression levels of the EF-hand calcium (Ca(2+)) sensor protein S100A1 has emerged as a key factor in reconstituting normal Ca(2+) handling in failing myocardium. Improved sarcoplasmic reticulum (SR) function with enhanced Ca(2+) resequestration appears critical for S100A1's cyclic adenosine monophosphate-independent inotropic effects but raises concerns about potential diastolic SR Ca(2+) leakage that might trigger fatal arrhythmias. This study shows for the first time a diminished interaction between S100A1 and ryanodine receptors (RyR2s) in experimental HF. Restoring this link in failing cardiomyocytes, engineered heart tissue and mouse hearts, respectively, by means of adenoviral and adeno-associated viral S100A1 cDNA delivery normalizes diastolic RyR2 function and protects against Ca(2+)- and ß-adrenergic receptor-triggered proarrhythmogenic SR Ca(2+) leakage in vitro and in vivo. S100A1 inhibits diastolic SR Ca(2+) leakage despite aberrant RyR2 phosphorylation via protein kinase A and calmodulin-dependent kinase II and stoichiometry with accessory modulators such as calmodulin, FKBP12.6 or sorcin. Our findings demonstrate that S100A1 is a regulator of diastolic RyR2 activity and beneficially modulates diastolic RyR2 dysfunction. S100A1 interaction with the RyR2 is sufficient to protect against basal and catecholamine-triggered arrhythmic SR Ca(2+) leak in HF, combining antiarrhythmic potency with chronic inotropic actions.
Asunto(s)
Insuficiencia Cardíaca/genética , Canal Liberador de Calcio Receptor de Rianodina/genética , Proteínas S100/metabolismo , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Calmodulina/metabolismo , ADN Complementario/metabolismo , Electrocardiografía , Técnicas de Transferencia de Gen , Insuficiencia Cardíaca/prevención & control , Masculino , Ratones , Microscopía Fluorescente , Miocardio/metabolismo , Miocitos Cardíacos/citología , Fosforilación , Unión Proteica , Ratas , Ratas Sprague-Dawley , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Proteínas de Unión a Tacrolimus/metabolismo , Ingeniería de Tejidos/métodosRESUMEN
PURPOSE: Selective delivery of drugs into the target tissue is expected to result in high drug concentrations in the tissue of interest and therefore enhanced drug efficacy. To develop a peptide-based radiopharmaceutical, we investigated the properties of a peptide with affinity for human breast cancer, which has been selected through phage display. EXPERIMENTAL DESIGN: The bioactivity of the p160 peptide (VPWMEPAYQRFL) was evaluated in vitro and in vivo. The specific binding to human breast cancer MDA-MB-435 cells was confirmed in competition experiments. Internalization of the peptide was investigated with confocal microscopy. Furthermore, the biodistribution of (131)I-labeled p160 was studied in tumor-bearing mice. In vivo stability was evaluated at different periods after tracer administration using high-performance liquid chromatography analysis. RESULTS: The binding of (125)I-labeled p160 was inhibited up to 95% by the unlabeled peptide with an IC(50) value of 0.6 micromol/L. In addition, 40% of the total bound activity was found to be internalized into the human breast cancer cells. Although a rapid degradation was seen, biodistribution studies in nude mice showed a higher uptake in tumor than in most of the organs. Perfusion of the animals caused a reduction of the radioligand accumulation in the healthy tissues, whereas the tumor uptake remained constant. A comparison of [(131)I]p160 with a (131)I-labeled Arg-Gly-Asp peptide revealed a higher tumor-to-organ ratio for [(131)I]p160. CONCLUSIONS: p160 has properties that make it an attractive carrier for tumor imaging and the intracellular delivery of isotopes or chemotherapeutic drugs.