RESUMO
High-fidelity genetically encoded bio-sensors that respond to changes in cellular environmental milieu in disease offer great potential in a range of patho-physiological settings. Here a unique hypoxia-regulated vector-based system with double oxygen-sensing transcriptional elements was developed for rapid and robust hypoxia-regulated gene expression in the heart. Hypoxia-responsive cis elements were used in tandem with a single proline-modified oxygen-dependent degradation (ODD) domain of hypoxia-inducible factor-1alpha to form a double oxygen-sensing vector system (DOSVS). In adult cardiac myocytes in vitro, the DOSVS demonstrated a low background expression not different from baseline control in normoxia, and with 100% efficiency, robust, 1,000-fold induction upon hypoxia. In the heart in vivo, hypoxic and ischemic challenges elicited rapid 700-fold induction in living animals, exceeding that obtained by a high-fidelity constitutive cytomegalovirus (CMV) viral promoter. DOSVS also showed high temporal resolution in the heart in response to cyclical bouts of hypoxia in vivo. We propose that DOSVS will be valuable for a range of applications, including bio-sensing and therapeutic gene expression in the heart and other organ systems that are confronted by chronic or episodic hypoxic/ischemic stresses in vivo.
Assuntos
Vetores Genéticos , Subunidade alfa do Fator 1 Induzível por Hipóxia/uso terapêutico , Hipóxia/terapia , Isquemia/terapia , Miocárdio/metabolismo , Oxigênio/metabolismo , Transdução Genética , Animais , Células Cultivadas , Citomegalovirus/genética , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Humanos , Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Técnicas In Vitro , Isquemia/genética , Luciferases/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Regiões Promotoras Genéticas/genética , Ratos , Ratos Sprague-Dawley , Elementos de Resposta/genética , Ativação TranscricionalRESUMO
The myofilament protein troponin I (TnI) has a key isoform-dependent role in the development of contractile failure during acidosis and ischemia. Here we show that cardiac performance in vitro and in vivo is enhanced when a single histidine residue present in the fetal cardiac TnI isoform is substituted into the adult cardiac TnI isoform at codon 164. The most marked effects are observed under the acute challenges of acidosis, hypoxia, ischemia and ischemia-reperfusion, in chronic heart failure in transgenic mice and in myocytes from failing human hearts. In the isolated heart, histidine-modified TnI improves systolic and diastolic function and mitigates reperfusion-associated ventricular arrhythmias. Cardiac performance is markedly enhanced in transgenic hearts during reperfusion despite a high-energy phosphate content similar to that in nontransgenic hearts, providing evidence for greater energetic economy. This pH-sensitive 'histidine button' engineered in TnI produces a titratable molecular switch that 'senses' changes in the intracellular milieu of the cardiac myocyte and responds by preferentially augmenting acute and long-term function under pathophysiological conditions. Myofilament-based inotropy may represent a therapeutic avenue to improve myocardial performance in the ischemic and failing heart.
