Khurram, Obaid U.

Neuromotor control of respiratory muscles such as the diaphragm muscle (DIAm), the main inspiratory muscle in mammals, is organized around motor units. Motor units comprise a motoneuron and the muscle fibers they innervate. Diversity in the contractile and fatigue properties of DIAm motor units permits generation of the wide range of behaviors necessary to sustain ventilation and maintain airways patent (e.g., sighs, which generate ~70% of maximal force in rats). Descending inputs to phrenic motoneurons innervating the DIAm is thought to be primarily unilateral in rats. After disrupting ipsilateral inputs by unilateral spinal cord hemisection at C2 (C2SH), latent, bilateral projections underlie recovery of ipsilateral DIAm activity. We hypothesized that DIAm EMG activity reflects bilateral descending premotor drive to motoneurons that varies across motor behaviors and conditions (anesthetized vs. awake). Using chronic DIAm EMG recordings in adult male Sprague‐Dawley rats, the bilateral vs. ipsilateral nature of premotor drive for each behavior and condition was evaluated by comparing the impact of C2SH. Three days prior to the C2SH surgery, a pair of DIAm electrodes was implanted bilaterally and DIAm EMG activity was assessed immediately prior to and after C2SH as well as at 3 days post‐C2SH in spontaneously‐breathing awake, restrained animals and following induction of ketamine‐xylazine anesthesia. DIAm EMG recordings comprised ~5 min of eupnea and exposure to hypoxia (10% O 2 )‐hypercapnia (5% CO 2 ). The completeness of C2SH surgery was confirmed by the absence of ipsilateral diaphragm EMG activity during eupnea at the time of C2SH surgery and at 3 days post‐C2SH in anesthetized animals, consistent with previous studies. In addition, histological assessment of disruption of anterolateral white matter tracts was conducted to verify the C2SH injury. Ipsilateral RMS EMG amplitude in anesthetized rats pre‐C2SH was ~30% and 45% of pre‐injury anesthetized sigh levels during eupnea and hypoxia‐hypercapnia, respectively. During awake conditions pre‐C2SH, ipsilateral RMS EMG amplitude was significantly higher (~50%, 70% and 200% of pre injury anesthetized sigh levels during eupnea, hypoxia‐hypercapnia and sighs, respectively). There were no differences across diaphragm muscle sides pre‐C2SH in RMS EMG amplitude. At 3 days post‐C2SH, ipsilateral RMS EMG activity was evident in anesthetized animals during hypoxia‐hypercapnia and sighs (~10% and 50% of pre‐injury anesthetized sigh levels) and in awake animals during eupnea, hypoxia‐hypercapnia and sighs (~25%, 50% and 125% of pre‐injury anesthetized sigh levels, respectively). These findings represent considerable differences in DIAm activity across motor behaviors and anesthetized vs. awake conditions and highlight physiologically‐relevant differences in the relative contribution of ipsilateral vs. bilateral inputs to phrenic motoneurons. Support or Funding Information NIH grant R01 HL96750.

We characterized three phases of neuromotor control—motor unit recruitment, sustained activity, and derecruitment—based on statistical assessments of stationarity of the diaphragm muscle (DIAm) EMG activity in awake rats. Our findings may allow indirect estimates of the contribution of motor unit recruitment and frequency coding toward generating force and provide novel insights about the temporal aspects of DIAm neuromotor control and descending respiratory drive in unanesthetized animals.

Recent findings suggest that autophagy plays a role in the development of aging-related neuromuscular dysfunction; however, the contribution of autophagy impairment to the maintenance of diaphragm force generation in old age is unknown. This study shows that in old mice, chloroquine administration decreases maximal transdiaphragmatic pressure generation. These chloroquine effects suggest a susceptibility to impairments in autophagy in old age.

Transdiaphragmatic pressure (Pdi) measurements are used to determine diaphragm force generation across various motor behaviors. Previous studies in the diaphragm muscle found selective age-related changes at fast-twitch fatigable motor units including increased susceptibility to neuromuscular transmission failure and denervation. Accordingly, age-related changes at diaphragm motor units predominantly impair the ability to accomplish higher force behaviors, and these aging effects are evident prior to the onset of muscle atrophy. Recent findings suggest that autophagy plays a critical role in the development of age-related neuromuscular dysfunction; however, the possible contribution of autophagy impairment to the maintenance of diaphragm force generation in old age is unknown. We hypothesized that inhibition of autophagy (using chloroquine) decreases diaphragm force-generating capacity (Pdi) in mice susceptible to age-related neuromuscular dysfunction. Adult male and female C57BL/6 mice at 16-28 months of age were randomized to receive a single intraperitoneal injection of 50 mg/kg of chloroquine or vehicle (n= 9 per treatment group) four hours before Pdi measurements, with the experimenter blinded. Pdi was measured during eupnea (room air breathing), exposure to hypoxia/hypercapnia (10% O2/5% CO2), tracheal occlusion, spontaneous deep breaths (“sighs”) and during maximal activation elicited by bilateral phrenic nerve stimulation. Consistent with our hypothesis, there was an ~25% reduction in maximal Pdi in the chloroquine group (46 ± 12 cm H2O) compared to the vehicle control (61 ± 16 cm H2O; p < 0.05). There was no effect of chloroquine on Pdi during eupnea, hypoxia/hypercapnia, tracheal occlusion, or sigh behaviors. Acute inhibition of autophagy results in reduced maximal Pdi, consistent with that evident in old mice. Thus, we propose that autophagy impairment may be an important contributor to the reduced diaphragm force generation associated with aging. These findings help us further understand the mechanisms regulating age-related neuromuscular dysfunction and may serve as a foundation to develop novel treatments to mitigate aging effects. Support: NIH R01 AG057052. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The diaphragm muscle is the primary inspiratory muscle in mammals and is highly active throughout life. The repetitive activation of the diaphragm muscle (and of other muscles driven by central pattern generator activity) presents an opportunity to analyze these physiological data on a per‐event basis. Here we highlight the development and implementation of the Automated Evaluation of Respiratory Signals (AERS) algorithm to detect individual respiratory events across a range of motor behaviors. Because different respiratory‐related signals provide different types of information, the AERS algorithm is designed to be robust regardless of the signal type—e.g., diaphragm electromyography (EMG) and transdiaphragmatic pressure (P di ). Individual respiratory event data obtained from adult rats were assessed using the AERS algorithm. This analysis revealed diaphragm root‐mean‐square (RMS) EMG and Pdi were lowest during eupnea, increased slightly in response to hypoxia‐hypercapnia (10% O 2 ‐ 5% CO 2 ), and increased approximately 3‐fold in response to airway occlusion compared to the eupneic baseline. The coefficient of variation (CV) within subjects was less than 10% across all behaviors for both RMS EMG and Pdi. By contrast, the CV was approximately 30% across subjects. Respiratory rate was increased by ~20 breaths/min during hypoxia‐hypercapnia compared to eupnea, as expected based on previous work from our lab and others. The respiratory rate CV within subjects was less than 10% during eupnea and hypoxia‐hypercapnia; during airway occlusion, the CV was ~17%, likely owing to the small number of respiratory events (2‐5) analyzed for this behavior per animal. For eupnea and hypoxia‐hypercapnia, the CV across subjects was ~20%, whereas during airway occlusion it was ~25%. Overall, our findings validate the mean values across various measures of respiratory motor output compared to standard subject means approach. However, per‐event analyses greatly enhance statistical power, allowing detection of physiologically relevant measures by taking within‐subject variability into account. Additionally, the AERS analyses show that various measures of respiratory variability as well as time‐frequency analyses can provide deeper insights into respiratory drive when accounting for individual respiratory events. These analyses can be effectively applied to a variety of models of respiratory motor unit loss or dysfunction (e.g., spinal cord injury and aging), allowing the extraction of large amounts of data from respiratory signals for more comprehensive assessments. Our findings suggest that the AERS approach may be particularly beneficial in reducing animal numbers in certain types of studies, for the detection of physiological changes reflected in respiratory signals with interventions causing gain or loss of function, and for analyses of respiratory variability, where breath‐to‐breath dynamics are important.

Motor units are normally recruited in an orderly manner in order to accomplish ventilatory behaviors. The duration of non‐stationarity in electromyographic (EMG) bursts provides an estimate of the period of motor unit recruitment. Motor unit recruitment also reflects central drive, which can be estimated by the root‐mean‐squared (RMS) EMG value 75 ms after the EMG burst onset (RMS 75 ). Following unilateral diaphragm muscle (DIAm) paralysis induced by upper cervical spinal cord hemisection at C 2 (SH), there is gradual spontaneous recovery of ipsilateral EMG activity. Increased activity of the contralateral (uninjured) DIAm is sufficient to maintain ventilation following SH. We hypothesized that changes in central drive can be estimated by the period of non‐stationarity and RMS 75 from the contralateral DIAm EMG following ipsilateral paralysis and spontaneous recovery of DIAm EMG activity. We found that the duration of non‐stationarity increased to a greater extent in rats that did not recover ipsilateral EMG activity by 14 days post‐SH (D14) compared to those that did. In addition, we found that RMS 75 values following SH were reduced in rats displaying recovery of ipsilateral DIAm EMG activity compared to rats that did not. Collectively, these results suggest that the duration of non‐stationarity and RMS 75 can be used to evaluate recovery of ipsilateral DIAm function following spinal cord injury.

The diaphragm muscle (DIAm) is the primary inspiratory muscle responsible for force generation across a range of ventilatory and higher force airway clearance behaviors throughout life. Our laboratory has previously reported DIAm sarcopenia, the age‐related atrophy and weakening of skeletal muscle, in multiple rodent models. Interestingly, in Fischer 344 rats, there is an increase in body mass from 6 to 24 months of age, which is greater in females (~140%) than in males (~110%), suggesting potential sex‐related differences in the manifestation of sarcopenia. Transdiaphragmatic pressure (Pdi) measurements provide information about force generation in vivo across a range of motor behaviors. The purpose of this study was to evaluate if there are sex‐related differences in DIAm sarcopenia, and if such differences translate to a functional impact on Pdi generation. We hypothesized that because of differences in weight gain, DIAm sarcopenia is more pronounced in male vs. female Fischer 344 rats, and that there is a greater impact of sarcopenia on maximum Pdi generated by bilateral phrenic nerve stimulation (Pdi max ). Since Pdi generated across a range of ventilatory motor behaviors only requires <50% Pdi max , we also hypothesize that sarcopenia does not impair Pdi generation during ventilatory behaviors. Compared to 6‐month old rats, DIAm sarcopenia was apparent in ‐ 24‐month old rats of both sexes with a 30% reduction in maximum specific force. There was also a ~20% decrease in the cross‐sectional area of type IIx and/or IIb fibers with age, and this reduction was more pronounced in male rats. Thus, these mixed results do not entirely support our hypothesis of more pronounced DIAm sarcopenia in male rats. However, DIAm sarcopenia was associated with a ~30% decrease in Pdi max that was comparable between male and female rats. Importantly, the Pdi generated during ventilatory behaviors was unaffected by DIAm sarcopenia, even during more forceful ventilatory efforts induced by complete oropharyngeal airway occlusion. Although ventilatory behaviors are preserved with aging, a reduction in Pdi max indicates an impaired ability of the DIAm to generate higher force expulsive airway clearance behaviors necessary for maintaining airway patency. Support or Funding Information NIH R01‐AG044615 and T32‐HL105355 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Mid‐cervical spinal cord contusion injury results in tissue damage, disruption of spinal pathways, and motoneuron loss. Phrenic motoneurons located in C3–5 segments of the cervical spinal cord innervate the diaphragm muscle (DIAm), and unilateral C4 contusion results in loss of 40–50% of motoneurons ipsilateral to the injury (~25% of the total motoneuron pool). Over time after unilateral C4 spinal cord contusion injury, DIAm electromyography (EMG) increases both contralateral and ipsilateral to the side of injury, suggesting compensation due to an increased activation of the surviving motoneurons. However, whether C4 contusion impairs the ability of the DIAm to accomplish higher force motor behaviors is unknown. Transdiaphragmatic pressure (Pdi) was measured across motor behaviors over time after unilateral C4 contusion injury. Maximum Pdi (Pdi max ) was elicited by bilateral phrenic nerve stimulation at 7 days post‐injury. We hypothesized that Pdi max is reduced following C4 mid‐cervical contusion injury, which will constrain the Pdi generated during different motor behaviors. Since ventilatory behaviors of the DIAm require <50% Pdi max , we further hypothesized that Pdi generated during ventilatory behaviors of the DIAm is not impaired after unilateral C4 mid‐cervical spinal cord contusion injury. In support of our hypothesis, we observed that Pdi max was reduced by ~25% after C4 mid‐cervical spinal cord contusion injury compared to a laminectomy control group. This decrease in Pdi max is consistent with the extent of phrenic motoneuron loss following contusion injury. We also found that during both eupnea (quiet breathing) and breathing stimulated by 10% O 2 (hypoxia) and 5% CO 2 (hypercapnia), Pdi generation was unimpaired by C4 mid‐cervical spinal cord contusion injury, again consistent with the lower force requirement of these ventilatory motor behaviors. Prior to injury, Pdi generated during airway occlusion was ~40% of Pdi max . One day following contusion injury, the Pdi amplitude during airway occlusion was reduced from ~30 cm H 2 O to ~20 cm, but this reduction was completely reversed by 7 days post‐injury. The reduction in Pdi amplitude at one day post‐injury cannot be attributed to the ~25% loss of phrenic motoneurons, and thus, may reflect a disruption of input to phrenic motoneurons or acute inflammation of their surrounding milieu. Over time after injury, changes in the balance between inhibition and excitation may result in recovery of higher‐force behaviors. Support or Funding Information Funded by NIH R01‐HL096750 and T32‐HL105355 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

The diaphragm (DIA) muscle, which separates the abdominal and thoracic cavities, is the primary inspiratory muscle in mammals. Both transdiaphragmatic pressure (Pdi) and DIA electromyography (EMG) are used to assess respiratory activity across motor behaviors of varying levels of force. However, due to the proximity of the heart to the DIA, cardiac electrical activity can severely impact the quality of the EMG signal, especially in real‐time settings when cardiac activity cannot be removed. Classically, either boxcar functions of root mean square (RMS) type calculations or low‐pass filters are used to quantify DIA EMG activity; however, these techniques have the drawback of being highly influenced by cardiac noise, which occurs approximately four times more frequently than DIA EMG activity during quiet breathing (eupnea). In humans with surface DIA EMG, fixed sample entropy (fSE)—a tool for assessing the complexity of a time series—has been proposed as a method to quantify the intensity of DIA EMG activity across varying levels of muscle pressure generation. The parameters of fSE can be adjusted to be resistant to relatively slow changes in a signal resulting from cardiac activity while being highly susceptible to the relatively quick changes in motor unit action potentials in the time domain. Unsurprisingly, cardiac noise is a problem in non‐human animal studies as well, particularly in disease models when the DIA EMG activity levels are reduced. In the present study, we assessed the effectiveness of fSE in accurately reflecting changes in EMG activity in healthy rats with in‐dwelling DIA EMG electrodes. We hypothesized that fSE EMG would be resistant to changes in the signal caused by cardiac noise while reflecting changes caused by DIA EMG. We evaluated 24 different parameter settings for fSE assessments to optimize the correlation coefficient between the fSE and Pdi signals. In addition, we analyzed the RMS, the average rectified value (ARV), and the fSE of DIA EMG events during eupnea, exposure to an hypercapnic (5% CO 2 )‐hypoxic (10% O 2 ) environment, and during airway occlusion by performing detailed comparisons of individual respiratory events. In total, approximately 900 individual respiratory events per analysis technique were assessed across all subjects. Our results show that fSE EMG is extremely effective at eliminating cardiac noise and has a high correlation coefficient with the Pdi signal (.92). In contrast, the correlation coefficient between RMS EMG and Pdi is 0.83 and the correlation coefficient between ARV EMG and Pdi is 0.88, likely due to the high level of cardiac noise in the DIA EMG. Additionally, we show that ventilatory parameters such as instantaneous burst duration and respiratory frequency are similar between Pdi, RMS, ARV, and fSE EMG. These findings suggest that fSE is a reliable and effective alternative to using traditional RMS and ARV EMG techniques to estimate the magnitude of respiratory muscle electrical activity in muscles that are susceptible to cardiac noise.