RESUMEN
Troponin I (TnI) is a muscle-specific protein and plays an allosteric function in the Ca(2+) regulation of cardiac and skeletal muscle contraction. Expression of cloned cDNA in Escherichia coli is an essential approach to preparing human TnI and mutants for structural and functional studies. The expression level of cardiac TnI in E. coli is very low. To reduce the potential toxicity of cardiac TnI to the host cell, we constructed a bi-cistronic expression vector to co-express cardiac TnI and cardiac/slow troponin C (TnC), a natural binding partner of TnI and a protein that readily expresses in E. coli at high levels. The co-expression moderately increased the expression of cardiac TnI although a high amount of TnC protein was produced from the bi-cistronic mRNA. The use of an E. coli strain containing additional tRNAs for certain low bacterial usage eukaryotic codons improved the expression of cardiac TnI. Modifications of two 5'-regional codons that have predicted low usages in bacterial cells did not reproduce the improvement, indicating that not the 5' but the overall codon usage restricts the translational efficiency of cardiac TnI mRNA in E. coli. However, deletion of the cardiac TnI-specific N-terminal 28 amino acids significantly improved the protein expression independent of the host cell tRNA modifications. The results suggest that the regulatory N-terminal domain of cardiac TnI is a dominant factor for the incompatibility in bacterial cells, supporting its role in modulating the overall molecular conformation.
Asunto(s)
Escherichia coli/genética , Miocardio/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/toxicidad , Eliminación de Secuencia/genética , Troponina I/biosíntesis , Troponina I/genética , Adulto , Animales , Clonación Molecular/métodos , Regulación Bacteriana de la Expresión Génica , Vectores Genéticos/biosíntesis , Vectores Genéticos/genética , Humanos , Ratones , Fragmentos de Péptidos/biosíntesis , Conformación Proteica , Estructura Terciaria de Proteína/genética , Troponina C/biosíntesis , Troponina C/genética , Troponina I/toxicidadRESUMEN
BACKGROUND: Cardiac troponins in blood are the most preferred markers of myocardial damage. The fact that they are normally not found in the circulation provides a high level of clinical sensitivity and specificity even when cardiac lesions are small. After myocardial injury, the troponins enter the circulation, where they can be used for diagnosis of acute coronary syndromes. Thus, the cardiac troponins are paramount for disease classification and risk stratification. However, little is known about the long-term effects of the released troponins on cardiac function. METHODS AND RESULTS: In this study we prepared recombinant murine cardiac troponin I (mc-TnI) and murine cardiac troponin T and used them to immunize mice. We report that A/J mice immunized with mc-TnI developed severe inflammation of the myocardium with increased expression of inflammatory chemokines RANTES (regulated on activation normal T cell expressed and secreted), monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2, T-cell activation gene 3, and eotaxin and chemokine receptors CCR1, CCR2, and CCR5. The inflammation was followed by cardiomegaly, fibrosis, reduced fractional shortening, and 30% mortality over 270 days. In contrast, mice immunized with murine cardiac troponin T or with the control buffer showed little or no inflammation and no death. Furthermore, we demonstrate that mice preimmunized with mc-TnI before left anterior descending coronary artery ligation showed greater infarct size, more fibrosis, higher inflammation score, and reduced fractional shortening. CONCLUSIONS: Overall, our results show for the first time that provocation of an autoimmune response to mc-TnI induces severe inflammation in the myocardium followed by fibrosis and heart failure with increased mortality in mice.