The Association between the Change in Directly Measured Cardiorespiratory Fitness across Time and Mortality Risk.

BACKGROUND: The relationship between cardiorespiratory fitness (CRF) and mortality risk has typically been assessed using a single measurement, though some evidence suggests the change in CRF over time influences risk. This evidence is predominantly based on studies using estimated CRF (CRFe). The strength of this relationship using change in directly measured CRF over time in apparently healthy men and women is not well understood. PURPOSE: To examine the association of change in CRF over time, measured using cardiopulmonary exercise testing (CPX), with all-cause and disease-specific mortality and to compare baseline and subsequent CRF measurements as predictors of all-cause mortality. METHODS: Participants included 833 apparently healthy men and women (42.9 ±â€¯10.8 years) who underwent two maximal CPXs, the second CPX being ≥1 year following the baseline assessment (mean 8.6 years, range 1.0 to 40.3 years). Participants were followed for up to 17.7 (SD 11.8) years for all-cause-, cardiovascular disease- (CVD), and cancer mortality. Cox-proportional hazard models were performed to determine the association between the change in CRF, computed as visit 1 (CPX1) peak oxygen consumption (VO2peak [mL·kg-1·min-1]) - visit 2 (CPX2) VO2peak, and mortality outcomes. A Wald-Chi square test of equality was used to compare the strength of CPX1 to CPX2 VO2peak in predicting mortality. RESULTS: During follow-up, 172 participants died. Overall, the change in CPX-CRF was inversely related to all-cause, CVD, and cancer mortality (p < 0.05). Each 1 mL·kg-1·min-1 increase was associated with a ~11, 15, and 16% (all p < 0.001) reduction in all-cause, CVD, and cancer mortality, respectively. The inverse relationship between CRF and all-cause mortality was significant (p < 0.05) when men and women were examined independently, after adjusting for years since first CPX, baseline VO2peak, and age. Further, the Wald Chi-square test of equality found CPX2 VO2peak to be a significantly stronger predictor of all-cause mortality than CPX1 VO2peak (p < 0.05). CONCLUSION: The change in CRF over time was inversely related to mortality outcomes, and mortality was better predicted by CRF measured at subsequent test than CPX1 CRF. These findings emphasize the importance of adopting lifestyle behaviors that promote CRF, as well as support the need for routine assessment of CRF in clinical practice to better assess risk.

Cardiorespiratory Fitness and Mortality in Healthy Men and Women.

BACKGROUND: There is a well-established inverse relationship between cardiorespiratory fitness (CRF) and mortality. However, this relationship has almost exclusively been studied using estimated CRF. OBJECTIVES: This study aimed to assess the association of directly measured CRF, obtained using cardiopulmonary exercise (CPX) testing with all-cause, cardiovascular disease (CVD), and cancer mortality in apparently healthy men and women. METHODS: Participants included 4,137 self-referred apparently healthy adults (2,326 men, 1,811 women; mean age: 42.8 ± 12.2 years) who underwent CPX testing to determine baseline CRF. Participants were followed for 24.2 ± 11.7 years (1.1 to 49.3 years) for mortality. Cox-proportional hazard models were performed to determine the relationship of CRF (ml·kg-1·min-1) and CRF level (low, moderate, and high) with mortality outcomes. RESULTS: During follow-up, 727 participants died (524 men, 203 women). CPX-derived CRF was inversely related to all-cause, CVD, and cancer mortality. Low CRF was associated with higher risk for all-cause (hazard ratio [HR]: 1.73; 95% confidence interval [CI]: 1.20 to 3.50), CVD (HR: 2.27; 95% CI: 1.20 to 3.49), and cancer (HR: 2.07; 95% CI: 1.18 to 3.36) mortality compared with high CRF. Further, each metabolic equivalent increment increase in CRF was associated with a 11.6%, 16.1%, and 14.0% reductions in all-cause, CVD, and cancer mortality, respectively. CONCLUSIONS: Given the prognostic ability of CPX-derived CRF for all-cause and disease-specific mortality outcomes, its use should be highly considered for apparently healthy populations as it may help to improve the efficacy of the individualized patient risk assessment and guide clinical decisions.

Branch-Specific Microtubule Destabilization Mediates Axon Branch Loss during Neuromuscular Synapse Elimination.

Developmental axon remodeling is characterized by the selective removal of branches from axon arbors. The mechanisms that underlie such branch loss are largely unknown. Additionally, how neuronal resources are specifically assigned to the branches of remodeling arbors is not understood. Here we show that axon branch loss at the developing mouse neuromuscular junction is mediated by branch-specific microtubule severing, which results in local disassembly of the microtubule cytoskeleton and loss of axonal transport in branches that will subsequently dismantle. Accordingly, pharmacological microtubule stabilization delays neuromuscular synapse elimination. This branch-specific disassembly of the cytoskeleton appears to be mediated by the microtubule-severing enzyme spastin, which is dysfunctional in some forms of upper motor neuron disease. Our results demonstrate a physiological role for a neurodegeneration-associated modulator of the cytoskeleton, reveal unexpected cell biology of branch-specific axon plasticity and underscore the mechanistic similarities of axon loss in development and disease.

Small Molecule Inhibitors Limit Endothelial Cell Invasion by Staphylococcus aureus.

Staphylococcus aureus is a leading causative agent in sepsis, endocarditis, and pneumonia. An emerging concept is that prognosis worsens when the infecting S. aureus strain has the capacity to not only colonize tissue as an extracellular pathogen, but to invade host cells and establish intracellular bacterial populations. In previous work, we identified host CDC42 as a central regulator of endothelial cell invasion by S. aureus. In the current work, we report that ML 141, a first-in-class CDC42 inhibitor, decreases invasion and resultant pathogenesis in a dose-dependent and reversible manner. Inhibition was found to be due in part to decreased remodeling of actin that potentially drives endocytic uptake of bacteria/fibronectin/integrin complexes. ML 141 decreased binding to fibronectin at these complexes, thereby limiting a key pathogenic mechanism used by S. aureus to invade. Structural analogs of ML 141 were synthesized (designated as the RSM series) and a subset identified that inhibit invasion through non-cytotoxic and non-bactericidal mechanisms. Our results support the development of adjunctive therapeutics targeting host CDC42 for mitigating invasive infection at the level of the host.

Lysosomal activity associated with developmental axon pruning.

Clearance of cellular debris is a critical feature of the developing nervous system, as evidenced by the severe neurological consequences of lysosomal storage diseases in children. An important developmental process, which generates considerable cellular debris, is synapse elimination, in which many axonal branches are pruned. The fate of these pruned branches is not known. Here, we investigate the role of lysosomal activity in neurons and glia in the removal of axon branches during early postnatal life. Using a probe for lysosomal activity, we observed robust staining associated with retreating motor axons. Lysosomal function was involved in axon removal because retreating axons were cleared more slowly in a mouse model of a lysosomal storage disease. In addition, we found lysosomal activity in the cerebellum at the time of, and at sites where, climbing fibers are eliminated. We propose that lysosomal activity is a central feature of synapse elimination. Moreover, staining for lysosomal activity may serve as a marker for regions of the developing nervous system undergoing axon pruning.

Simvastatin inhibits Staphylococcus aureus host cell invasion through modulation of isoprenoid intermediates.

Patients on a statin regimen have a decreased risk of death due to bacterial sepsis. We have found that protection by simvastatin includes the inhibition of host cell invasion by Staphylococcus aureus, the most common etiologic agent of sepsis. Inhibition was due in part to depletion of isoprenoid intermediates within the cholesterol biosynthesis pathway and led to the cytosolic accumulation of the small GTPases CDC42, Rac, and RhoB. Actin stress fiber disassembly required for host invasion was attenuated by simvastatin and by the inhibition of phosphoinositide 3-kinase (PI3K) activity. PI3K relies on coupling to prenylated proteins, such as this subset of small GTPases, for access to membrane-bound phosphoinositide to mediate stress fiber disassembly. Therefore, we examined whether simvastatin restricts PI3K cellular localization. In response to simvastatin, the PI3K isoform p85, coupled to these small-GTPases, was sequestered within the cytosol. From these findings, we propose a mechanism whereby simvastatin restricts p85 localization, inhibiting the actin dynamics required for bacterial endocytosis. This approach may provide the basis for protection at the level of the host in invasive infections by S. aureus.

Axon branch removal at developing synapses by axosome shedding.

In many parts of the developing nervous system, the number of axonal inputs to each postsynaptic cell is dramatically reduced. This synapse elimination has been extensively studied at the neuromuscular junction, but how axons are lost is unknown. Here, we combine time-lapse imaging of fluorescently labeled axons and serial electron microscopy to show that axons at neuromuscular junctions are removed by an unusual cellular mechanism. As axons disappear, they shed numerous membrane bound remnants. These "axosomes" contain a high density of synaptic organelles and are formed by engulfment of axon tips by Schwann cells. After this engulfment, the axosome's contents mix with the cytoplasm of the glial cell. Axosome shedding might underlie other forms of axon loss and may provide a pathway for interactions between axons and glia.

Age-associated synapse elimination in mouse parasympathetic ganglia.

Little is known about the effects of aging on synapses in the mammalian nervous system. We examined the innervation of individual mouse submandibular ganglion (SMG) neurons for evidence of age-related changes in synapse efficacy and number. For approximately 85% of adult life expectancy (30 months) the efficacy of synaptic transmission, as determined by excitatory postsynaptic potential (EPSP) amplitudes, remains constant. Similarly, the number of synapses contacting individual SMG neurons is also unchanged. After 30 months of age, however, some neurons (23%) dramatically lose synaptic input exhibiting both smaller EPSP amplitude and fewer synaptic boutons. Attenuation of both the amplitude and frequency of miniature EPSPs was also observed in neurons from aged animals. Electron micrographs revealed that, although there were many vesicle-laden preganglionic axonal processes in the vicinity of the postsynaptic membrane, the number of synaptic contacts was significantly lower in old animals. These results demonstrate primary, age-associated synapse elimination with functional consequences that cannot be explained by pre- or postsynaptic cell death.

Ectopic endplates induce localized changes in skeletal muscle ultrastructure.

To investigate the processes by which motoneurons control protein synthesis, and thus the ultrastructure of the muscle fibers they innervate, ectopic endplates were induced to form on adult mouse skeletal muscle fibers by transplantation of a foreign nerve onto the muscle. In the dually innervated muscle fibers thus created, we examined two ultrastructural parameters that correlate with the expression of distinct isoforms of the myofibrillar proteins alpha-actinin and titin, specifically, Z-line width and sarcomere length. It was found that Z-lines were significantly thinner (98 vs. 128 nm) and sarcomeres were significantly shorter (1.69 vs. 2.06 microm) near the ectopic than near the original endplates. Thus, ectopic endplate formation on adult skeletal muscle fibers induces a localized alteration in myofibrillar morphology. These results may help to elucidate the role played by motoneurons in the determination and maintenance of muscle fiber properties and the processes that occur following muscle reinnervation after nerve injury.