RESUMO
Cardiomyopathy is a major cause of morbidity and mortality. Ventricular conduction delay, as shown by prolonged deflections in the electrocardiogram caused by delayed ventricular contraction (wide QRS complex), is a common feature of cardiomyopathy and is associated with a poor prognosis. Although the G(i)-signaling pathway is up-regulated in certain cardiomyopathies, previous studies suggested this up-regulation was compensatory rather than a potential cause of the disease. Using the tetracycline transactivator system and a modified G(i)-coupled receptor (Ro1), we provide evidence that increased G(i) signaling in mice can result in a lethal cardiomyopathy associated with a wide QRS complex arrhythmia. Induced expression of Ro1 in adult mice resulted in a >90% mortality rate at 16 wk, whereas suppression of Ro1 expression after 8 wk protected mice from further mortality and allowed partial improvement in systolic function. Results of DNA-array analysis of over 6,000 genes from hearts expressing Ro1 are consistent with hyperactive G(i) signaling. DNA-array analysis also identified known markers of cardiomyopathy and hundreds of previously unknown potential diagnostic markers and therapeutic targets for this syndrome. Our system allows cardiomyopathy to be induced and reversed in adult mice, providing an unprecedented opportunity to dissect the role of G(i) signaling in causing cardiac pathology.
Assuntos
Cardiomiopatias/fisiopatologia , Receptores Opioides kappa/fisiologia , Função Ventricular/fisiologia , Animais , Cardiomiopatias/genética , Doxiciclina/farmacologia , Eletrocardiografia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Coração/fisiopatologia , Camundongos , Camundongos Transgênicos , Miocárdio/patologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/fisiologia , Receptores Opioides kappa/genética , Transdução de Sinais , Análise de Sobrevida , Taquicardia Ventricular/genética , Taquicardia Ventricular/fisiopatologia , Resistência a Tetraciclina/genética , Função Ventricular/efeitos dos fármacos , Função Ventricular/genética , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Ligand-conjugated polymer (polyplex) gene delivery vectors have strong potential as targeted, in vivo gene transfer vehicles; however, they are currently limited by low delivery efficiency. A number of barriers to polyplex-mediated delivery have been previously identified, including receptor binding, internalization, endosomal escape, and nuclear localization. However, based on understanding of viral gene delivery systems, yet another potential barrier may exist; a limited ability to unpackage the plasmid DNA cargo following localization to the nucleus. We have developed a model system that employs a cationic polymer linked to epidermal growth factor (EGF) as a ligand to target delivery of plasmid DNA encoding the green fluorescent protein to mouse fibroblasts bearing the EGF receptor. Using fluorescence microscopy to simultaneously trace both the plasmid and polymer during gene delivery in combination with an in vitro transcription assay, we provide evidence that plasmid unpackaging can indeed be a limiting step for gene expression for sufficiently large polymer constructs. Short-term expression is significantly enhanced by using short polycations that dissociate from DNA more rapidly both in vitro and in vivo. Finally, we describe a thermodynamic model that supports these data by showing that shorter polycations can have a higher probability of dissociating from DNA. This work demonstrates that vector unpackaging should be added to the list of barriers to receptor-mediated polyplex gene delivery, thus providing an additional design principle for targeted synthetic delivery vehicles.
