RESUMO
Cardiac microtubules have recently been implicated in mechanical dysfunction during heart failure. However, systemic intolerance and non-cardiac effects of microtubule-depolymerizing compounds have made it challenging to determine the effect of microtubules on myocardial performance. Herein, we leverage recent advancements in living myocardial slices to develop a stable working preparation that recapitulates the complexity of diastole by including early and late phases of diastolic filling. To determine the effect of cardiac microtubule depolymerization on diastolic performance, myocardial slices were perfused with oxygenated media to maintain constant isometric twitch forces for more than 90 min. Force-length work loops were collected before and after 90 min of treatment with either DMSO (vehicle) or colchicine (microtubule depolymerizer). A trapezoidal stretch was added prior to the beginning of ventricular systole to mimic late-stage-diastolic filling driven by atrial systole. Force-length work loops were obtained at fixed preload and afterload, and tissue velocity was obtained during diastole as an analog to trans-mitral Doppler. In isometric twitches, microtubule destabilization accelerated force development, relaxation kinetics, and decreased end diastolic stiffness. In work loops, microtubule destabilization increased stroke length, myocardial output, accelerated isometric contraction and relaxation, and increased the amplitude of early filling. Taken together, these results indicate that the microtubule destabilizer colchicine can improve diastolic performance by accelerating isovolumic relaxation and early filling leading to increase in myocardial work output.
RESUMO
The alternative sigma factor σ54 has been shown to regulate the expression of a wide array of virulence-associated genes, as well as central metabolism, in bacterial pathogens. In Gram-positive organisms, the σ54 is commonly associated with carbon metabolism. In this study, we show that the Enterococcus faecalis alternative sigma factor σ54 (RpoN) and its cognate enhancer binding protein MptR are essential for mannose utilization and are primary contributors to glucose uptake through the Mpt phosphotransferase system. To gain further insight into how RpoN contributes to global transcriptional changes, we performed microarray transcriptional analysis of strain V583 and an isogenic rpoN mutant grown in a chemically defined medium with glucose as the sole carbon source. Transcripts of 340 genes were differentially affected in the rpoN mutant; the predicted functions of these genes mainly related to nutrient acquisition. These differentially expressed genes included those with predicted catabolite-responsive element (cre) sites, consistent with loss of repression by the major carbon catabolite repressor CcpA. To determine if the inability to efficiently metabolize glucose/mannose affected infection outcome, we utilized two distinct infection models. We found that the rpoN mutant is significantly attenuated in both rabbit endocarditis and murine catheter-associated urinary tract infection (CAUTI). Here, we examined a ccpA mutant in the CAUTI model and showed that the absence of carbon catabolite control also significantly attenuates bacterial tissue burden in this model. Our data highlight the contribution of central carbon metabolism to growth of E. faecalis at various sites of infection.IMPORTANCE Hospital-acquired infections account for 2 billion dollars annually in increased health care expenses and cause more than 100,000 deaths in the United States alone. Enterococci are the second leading cause of hospital-acquired infections. They form biofilms at surgical sites and are often associated with infections of the urinary tract following catheterization. Nutrient uptake and growth are key factors that influence their ability to cause disease. Our research identified a large set of genes that illuminate nutrient uptake pathways in enterococci. Perturbation of the metabolic circuit reduces virulence in a rabbit endocarditis model, as well as in catheter-associated urinary tract infection in mice. Targeting metabolic pathways that are important in infection may lead to new treatments against multidrug-resistant enterococcal infections.
