Sieck, Gary

AbstractThe diaphragm muscle comprises various types of motor units that are recruited in an orderly fashion governed by the intrinsic electrophysiological properties (membrane capacitance as a function of somal surface area) of phrenic motor neurons (PhMNs). Glutamate is the main excitatory neurotransmitter at PhMNs and acts primarily via fast acting AMPA and N‐methyl‐D‐aspartic acid (NMDA) receptors. Differences in receptor expression may also contribute to motor unit recruitment order. We used single cell, multiplex fluorescence in situ hybridization to determine glutamatergic receptor mRNA expression across PhMNs based on their somal surface area. In adult male and female rats (n = 9) PhMNs were retrogradely labeled for analyses (n = 453 neurons). Differences in the total number and density of mRNA transcripts were evident across PhMNs grouped into tertiles according to somal surface area. A ~ 25% higher density of AMPA (Gria2) and NMDA (Grin1) mRNA expression was evident in PhMNs in the lower tertile compared to the upper tertile. These smaller PhMNs likely comprise type S motor units that are recruited first to accomplish lower force, ventilatory behaviors. In contrast, larger PhMNs with lower volume densities of AMPA and NMDA mRNA expression presumably comprise type FInt and FF motor units that are recruited during higher force, expulsive behaviors. Furthermore, there was a significantly higher cytosolic NMDA mRNA expression in small PhMNs suggesting a more important role for NMDA‐mediated glutamatergic neurotransmission at smaller PhMNs. These results are consistent with the observed order of motor unit recruitment and suggest a role for glutamatergic receptors in support of this orderly recruitment. image Cover Image for this issue: doi: 10.1111/jnc.14747.

New Findings What is the central question of this study? Is the residual force generated by the diaphragm muscle after repeated activation reduced with sarcopenia, and is the residual force generated after fatiguing activation sufficient to sustain ventilatory behaviours of diaphragm muscle in young and old rats? What is the main finding and its importance? After diaphragm muscle fatigue, the residual specific force after 120 s of repeated stimulation was unaffected by ageing and was sufficient to accomplish ventilatory behaviours, but not expulsive manoeuvres (e.g. coughing). The inability to perform expulsive behaviours might underlie the increased susceptibility of older individuals to respiratory tract infections. Abstract Type IIx and/or IIb diaphragm muscle (DIAm) fibres make up more fatigable motor units that are more vulnerable to sarcopenia, i.e. age‐associated reductions of specific force and cross‐sectional area. In contrast, type I and IIa DIAm fibres form fatigue‐resistant motor units that are relatively unchanged with age. The fatigue resistance of the DIAm is assessed by normalizing the residual force generated after a period of repeated supramaximal stimulation (e.g. 120 s) to the initial maximal force. Given that sarcopenia primarily affects more fatigable DIAm motor units, apparent fatigue resistance improves with ageing. However, the central question is whether there is an ageing‐related difference in the residual force generated by the DIAm after repeated stimulation and whether this force is sufficient to sustain ventilatory behaviours of DIAm. In 6‐ and 24‐month‐old Fischer 344 rats, we assessed the loss of ex vivo DIAm force throughout 120 s of repeated supramaximal stimulation at 10, 40 and 75 Hz. We found that relative fatigue resistance improved in older rats at 40 and 75 Hz stimulation. Across all stimulation frequencies, DIAm residual force was unchanged with age (∼5 N cm −2 ). We conclude that ageing increases the relative contribution of type I and IIa fibres to DIAm force, with decreased contributions of type IIx and/or IIb fibres. The residual force generated by the DIAm after repeated stimulation is sufficient to accomplish ventilatory behaviours, regardless of age.

AbstractStructural plasticity in motoneurons may be influenced by activation history and motoneuron–muscle fiber interactions. The goal of this study was to examine the morphological adaptations of phrenic motoneurons following imposed motoneuron inactivity while controlling for diaphragm muscle inactivity. Well‐characterized rat models were used including unilateral C2 spinal hemisection (SH; ipsilateral phrenic motoneurons and diaphragm muscle are inactive) and tetrodotoxin phrenic nerve blockade (TTX; ipsilateral diaphragm muscle is paralyzed while phrenic motoneuron activity is preserved). We hypothesized that inactivity of phrenic motoneurons would result in a decrease in motoneuron size, consistent with a homeostatic increase in excitability. Phrenic motoneurons were retrogradely labeled by ipsilateral diaphragm muscle injection of fluorescent dextrans or cholera toxin subunit B. Following 2 weeks of diaphragm muscle paralysis, morphological parameters of labeled ipsilateral phrenic motoneurons were assessed quantitatively using fluorescence confocal microscopy. Compared to controls, phrenic motoneuron somal volumes and surface areas decreased with SH, but increased with TTX. Total phrenic motoneuron surface area was unchanged by SH, but increased with TTX. Dendritic surface area was estimated from primary dendrite diameter using a power equation obtained from three‐dimensional reconstructed phrenic motoneurons. Estimated dendritic surface area was not significantly different between control and SH, but increased with TTX. Similarly, TTX significantly increased total phrenic motoneuron surface area. These results suggest that ipsilateral phrenic motoneuron morphological adaptations are consistent with a normalization of motoneuron excitability following prolonged alterations in motoneuron activity. Phrenic motoneuron structural plasticity is likely more dependent on motoneuron activity (or descending input) than muscle fiber activity.

New Findings What is the central question of this study? Absence of hypothermia‐induced cardiac arrest is a strong predictor for a favourable outcome after rewarming. Nevertheless, detailed knowledge of preferences in organ blood flow during rewarming with spontaneous circulation is largely unknown. What is the main finding and its importance? In a porcine model of accidental hypothermia, we find, despite a significantly reduced cardiac output during rewarming, normal blood flow and O2 supply in vital organs owing to patency of adequate physiological compensatory responses. In critical care medicine, active rewarming must aim at supporting the spontaneous circulation and maintaining spontaneous autonomous vascular control. AbstractThe absence of hypothermia‐induced cardiac arrest is one of the strongest predictors for a favourable outcome after rewarming from accidental hypothermia. We studied temperature‐dependent changes in organ blood flow and O2 delivery () in a porcine model with spontaneous circulation during 3 h of hypothermia at 27°C followed by rewarming. Anaesthetized pigs (n = 16, weighing 20–29 kg) were randomly assigned to one of two groups: (i) hypothermia/rewarming (n = 10), immersion cooled to 27°C and maintained for 3 h before being rewarmed by pleural lavage; and (ii) time‐matched normothermic (38°C) control animals (n = 6), immersed for 6.5 h, the last 2 h with pleural lavage. Regional blood flow was measured using a neutron‐labelled microsphere technique. Simultaneous measurements of and O2 consumption () were made. During hypothermia, there was a reduction in organ blood flow, and . After rewarming, there was a 40% reduction in stroke volume and cardiac output, causing a global reduction in ; nevertheless, blood flow to the brain, heart, stomach and small intestine returned to prehypothermic values. Blood flow in the liver and kidneys was significantly reduced. Cerebral and returned to control values. After hypothermia and rewarming there is a significant lowering of owing to heart failure. However, compensatory mechanisms preserve O2 transport, blood flow and in most organs. Nevertheless, these results indicate that hypothermia‐induced heart failure requires therapeutic intervention.

New Findings What is the central question of this study? Will discontinued stimulation of isolated cardiomyocytes (asystole) during hypothermia mitigate hypothermia–rewarming‐induced cytosolic Ca2+ overload? What is the main finding and its importance? Mimicking asystole or hypothermic cardiac arrest by discontinued stimulation of cardiomyocytes during hypothermia resulted in normal contractile function after rewarming. This result suggests that asystole during severe hypothermia provides protection from hypothermia–rewarming‐induced contractile dysfunction in cardiomyocytes. AbstractAfter exposure of spontaneously beating hearts or electrically stimulated isolated cardiomyocytes to hypothermia–rewarming (H/R), cardiac dysfunction or alteration in excitation–contraction coupling, respectively, is a consequence. In contrast, hypothermic cardiac arrest, as routinely applied during cardiac surgery, will not impose any hazard to cardiac function after rewarming. We hypothesize that by maintaining asystole during H/R, cardiomyocytes will avoid Ca2+ overload attributable to the transient stimulation‐evoked elevation of [Ca2+]i and thus, H/R‐induced elevation of phosphorylated cardiac troponin I and reduced Ca2+ sensitivity after rewarming. To test this hypothesis, the aim of the study was to determine whether discontinued electrical stimulation (to imitate hypothermic cardiac arrest) versus stimulation during 3 h of H/R prevents disruption of excitation–contraction coupling in our established cardiomyocyte H/R model. Cytosolic Ca2+ and the contractile response (sarcomere length shortening) were measured using an IonOptix system, and the dynamic assessment of Ca2+ sensitivity of contraction was conducted using a phase‐loop plot. Cardiomyocytes were divided into three groups. Group 1 (time‐matched control) was continuously stimulated at 0.5 Hz for 3 h at 35°C. Group 2 was continuously stimulated during H/R at 0.5 Hz, whereas in group 3 stimulation was discontinued during H/R and thus the cells remained quiescent until the resumption of stimulation after rewarming. The results demonstrate that discontinued stimulation of cardiomyocytes during H/R, imitating hypothermic cardiac arrest during cardiac surgery, provides protection against H/R‐induced disruption of excitation–contraction coupling. We suggest that protective effects are caused by preventing the protein kinase A‐induced elevation of phosphorylated cardiac troponin I, which is a key mechanism to reduce myofilament Ca2+ sensitivity of contraction.

Breathing occurs without thought but is controlled by a complex neural network with a final output of phrenic motor neurons activating diaphragm muscle fibers (i.e., motor units). This review considers diaphragm motor unit organization and how they are controlled during breathing as well as during expulsive behaviors.

Lewis, Michael I., Thomas J. Lorusso, Wen-Zhi Zhan, and Gary C. Sieck. Interactive effects of denervation and malnutrition on diaphragm structure and function. J. Appl. Physiol. 81(5): 2165–2172, 1996.—The purpose of this study was to examine the interactive effects of unilateral denervation (DN) and prolonged malnutrition (MN) on the structure and function of the diaphragm muscle (Dia). Four groups of rats were studied: control (Con), MN, DN, and DN-MN. MN began 2 wk after DN and lasted 4 wk. In both the DN and DN-MN groups, the relative loss in Dia weight exceeded the relative change in body weight. Compared with the Con group, Dia specific force was reduced by ∼40% in both the DN and DN-MN groups but was unaffected in the MN group. Dia fatigue resistance improved in all experimental groups but to a greater extent in the DN and DN-MN groups. In both the DN and DN-MN groups, ∼50% of Dia fibers were classified as type IIc, whereas fiber type proportions did not change in the MN group. In the DN group, only type IIb/x fibers atrophied, whereas all fiber types atrophied in the MN and DN-MN groups. We conclude that in the DN-MN group the reduction in specific force combined with the reduction in total cross-sectional area of the muscle significantly curtails Dia force-generating capacity.