Journal of Physiology
Time course analysis of mechanical ventilation-induced diaphragm contractile muscle dysfunction in the rat
Controlled mechanical ventilation (CMV) plays a key role in triggering the impaired diaphragm muscle function and the concomitant delayed weaning from the respirator in critically ill intensive care unit (ICU) patients. To date, experimental and clinical studies have primarily focused on early effects on the diaphragm by CMV, or at specific time points. To improve our understanding of the mechanisms underlying the impaired diaphragm muscle function in response to mechanical ventilation, we have performed time-resolved analyses between 6 h and 14 days using an experimental rat ICU model allowing detailed studies of the diaphragm in response to long-term CMV. A rapid and early decline in maximum muscle fibre force and preceding muscle fibre atrophy was observed in the diaphragm in response to CMV, resulting in an 85% reduction in residual diaphragm fibre function after 9–14 days of CMV. A modest loss of contractile proteins was observed and linked to an early activation of the ubiquitin proteasome pathway, myosin:actin ratios were not affected and the transcriptional regulation of myosin isoforms did not show any dramatic changes during the observation period. Furthermore, small angle X-ray diffraction analyses demonstrate that myosin can bind to actin in an ATP-dependent manner even after 9–14 days of exposure to CMV. Thus, quantitative changes in muscle fibre size and contractile proteins are not the dominating factors underlying the dramatic decline in diaphragm muscle function in response to CMV, in contrast to earlier observations in limb muscles. The observed early loss of subsarcolemmal neuronal nitric oxide synthase activity, onset of oxidative stress, intracellular lipid accumulation and post-translational protein modifications strongly argue for significant qualitative changes in contractile proteins causing the severely impaired residual function in diaphragm fibres after long-term mechanical ventilation. For the first time, the present study demonstrates novel changes in the diaphragm structure/function and underlying mechanisms at the gene, protein and cellular levels in response to CMV at a high temporal resolution ranging from 6 h to 14 days.
Force generation in the muscle sarcomere is driven by the head domain of the myosin molecule extending from the thick filament to form cross-bridges with the actin-containing thin filament. Following attachment, a structural working stroke in the head pulls the thin filament towards the centre of the sarcomere, producing, under unloaded conditions, a filament sliding of ~11 nm. The mechanism of force generation by the myosin head depends on the relationship between cross-bridge force and movement, which is determined by compliances of the cross-bridge (Ccb) and filaments. By measuring the force dependence of the spacing of the high-order myosin- and actin-based X-ray reflections from sartorius muscles of Rana esculenta we find a combined filament compliance (Cf) of 13.1 ± 1.2 nm MPa–1, close to recent estimates from single fibre mechanics (12.8 ± 0.5 nm MPa–1). Ccb calculated using these estimates is 0.37 ± 0.12 nm pN–1, a value fully accounted for by the compliance of the myosin head domain, 0.38 ± 0.06 nm pN–1, obtained from the intensity changes of the 14.5 nm myosin-based X-ray reflection in response to 3 kHz oscillations imposed on single muscle fibres in rigor. Thus, a significant contribution to Ccb from the myosin tail that joins the head to the thick filament is excluded. The low Ccb value indicates that the myosin head generates isometric force by a small sub-step of the 11 nm stroke that drives filament sliding at low load. The implications of these results for the mechanism of force generation by myosins have general relevance for cardiac and non-muscle myosins as well as for skeletal muscle.
Hypometabolism and hypothermia in the rat model of endotoxic shock: independence of circulatory hypoxia
We tested the hypothesis that development of hypothermia instead of fever in endotoxic shock is consequential to hypoxia. Endotoxic shock was induced by bacterial lipopolysaccharide (LPS, 500 μg kg–1 i.v.) in rats at an ambient temperature of 22°C. A β3-adrenergic agonist known to activate metabolic heat production, CL316,243, was employed to evaluate whether thermogenic capacity could be impaired by the fall in oxygen delivery (O2) during endotoxic shock. This possibility was rejected as CL316,243 (0.15 mg kg–1 i.v.) evoked similar rises in oxygen consumption (VO2) in the presence and absence of endotoxic shock. Next, to investigate whether a less severe form of circulatory hypoxia could be triggering hypothermia, the circulating volume of LPS-injected rats was expanded using 6% hetastarch with the intention of improving tissue perfusion and alleviating hypoxia. This intervention attenuated not only the fall in arterial pressure induced by LPS, but also the associated falls in VO2 and body temperature. These effects, however, occurred independently of hypoxia, as they were not accompanied by any detectable changes in NAD+/NADH ratios. Further experimentation revealed that even the earliest drops in cardiac output and O2 during endotoxic shock did not precede the reduction in VO2 that brings about hypothermia. In fact, O2 and VO2 fell in such a synchrony that the O2/VO2 ratio remained unaffected. Only when hypothermia was prevented by exposure to a warm environment (30°C) did an imbalance in the O2/VO2 ratio become evident, and such an imbalance was associated with reductions in the renal and hypothalamic NAD+/NADH ratios. In conclusion, hypometabolism and hypothermia in endotoxic shock are not consequential to hypoxia but serve as a pre-emptive strategy to avoid hypoxia in this model.
Central command dysfunction in rats with heart failure is mediated by brain oxidative stress and normalized by exercise training
In heart failure, sympathoexcitation elicited by central command, a parallel activation of the motor and autonomic neural circuits in the brain, is exaggerated.
Mechanisms underlying central command dysfunction in heart failure were unexplored, and effects of exercise training on central command dysfunction in heart failure were not determined.
Data presented here suggest that oxidative stress in the medulla in heart failure mediates central command dysfunction, and that exercise training in heart failure is capable of normalizing central command dysfunction through its antioxidant effects in the medulla.
The present study contributes to our understanding of brain mechanisms underlying abnormal autonomic adjustments to exercise in heart failure.
Sympathoexcitation elicited by central command, a parallel activation of the motor and autonomic neural circuits in the brain, has been shown to become exaggerated in chronic heart failure (CHF). The present study tested the hypotheses that oxidative stress in the medulla in CHF plays a role in exaggerating central command-elicited sympathoexcitation, and that exercise training in CHF suppresses central command-elicited sympathoexcitation through its antioxidant effects in the medulla. In decerebrate rats, central command was activated by electrically stimulating the mesencephalic locomotor region (MLR) after neuromuscular blockade. The MLR stimulation at a current intensity greater than locomotion threshold in rats with CHF after myocardial infarction (MI) evoked larger (P < 0.05) increases in renal sympathetic nerve activity and arterial pressure than in sham-operated healthy rats (Sham) and rats with CHF that had completed longterm (8–12 weeks) exercise training (MI + TR). In the Sham and MI + TR rats, bilateral microinjection of a superoxide dismutase (SOD) mimetic Tempol into the rostral ventrolateral medulla (RVLM) had no effects on MLR stimulation-elicited responses. By contrast, in MI rats, Tempol treatment significantly reduced MLR stimulation-elicited responses. In a subset of MI rats, treatment with Tiron, another SOD mimetic, within the RVLM also reduced responses. Superoxide generation in the RVLM, as evaluated by dihydroethidium staining, was enhanced in MI rats compared with that in Sham and MI + TR rats. Collectively, these results support the study hypotheses. We suggest that oxidative stress in the medulla in CHF mediates central command dysfunction, and that exercise training in CHF is capable of normalizing central command dysfunction through its antioxidant effects in the medulla.