Sieck, Gary C.

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.

Sarcopenia is the aging‐related loss of force‐generating capacity and atrophy of skeletal muscles and is a major contributor to morbidity and mortality in the elderly. The molecular mechanisms underlying sarcopenia remain to be elucidated. Skeletal muscles exhibit different rates of sarcopenia, generally reflecting their specific muscle fiber type composition. Muscles primarily composed of type IIx and/or IIb muscle fibers (i.e., extensor digitorum longus; EDLm) develop sarcopenia at a faster rate than other muscles. In the diaphragm, muscle fiber cross‐sectional area (CSA) is reduced in old age, predominantly in type IIx and/or IIb fibers. Autophagy is a multistep, catabolic process that targets dysfunctional cytoplasmatic structures including proteins and organelles for degradation and recycling. Autophagy is impaired in old age across several tissues, including motor neurons and skeletal muscle. Expression of core autophagy proteins can indicate changes in autophagy, e.g., LC3 reflects initiation/elongation steps and p62 clearance reflects degradation. We hypothesize that impaired autophagy is associated with the development of sarcopenia in type‐identified muscle fibers of the EDLm. Indeed, at 24‐months of age, the CSA of type I and IIa fibers was not reduced, whereas the CSA of type IIx and/or IIb fibers was ~25% smaller (p<0.01 in both cases), compared to 6‐month‐old mice. These findings are consistent with fiber type selective sarcopenia in the EDLm. Expression of LC3 and p62 in the EDLm of mice at 6‐ and 24‐ months of age was evaluated via immunofluorescence by quantification of the number of LC3 and p62 puncta per muscle fiber type. In type I and IIa fibers, the number of LC3 puncta increased by ~30% and of p62 increased three‐fold in 24‐ compared to 6‐month‐old mice (p<0.01 in both cases). In type IIx and/or IIb fibers, the number of LC3 puncta decreased by ~60% and p62 puncta increased by ~50% in 24‐ compared to 6‐month‐old mice (p<0.01 in both cases). Accordingly, compared to 6 month‐old mice, autophagic degradation is impaired across muscle fiber types in 24‐month old mice, but likely to a greater extent in type I and IIa fibers of old mice given the increase in both LC3 and p62. Autophagic initiation is primarily reduced in type IIx and/or IIb fibers in the EDL as indicated by the reduced LC3 expression. Taken together these findings indicate that fiber type selective sarcopenia may reflect differences in autophagy across muscle fiber types.

Tumor necrosis factor α (TNFα) contributes to the pathophysiology of several inflammatory airway diseases such as asthma, chronic obstructive pulmonary disease (COPD), chronic bronchitis, and COVID‐19. Previously, we showed that acute (24‐h) exposure to TNFα promotes mitochondrial fragmentation in human airway smooth muscle (hASM) cells, which is associated with an increased level of cytosolic GTPase dynamin‐related protein 1 (DRP1), a major protein involved in mitochondrial fragmentation. Phosphorylation of DRP1 at Ser616 (S616) promotes its translocation and binding to the outer mitochondrial membrane and induces mitochondrial fragmentation; however, phosphorylation of DRP1 at the Ser637 (S637) residue reverses the total process and diminishes fragmentation. The balance between S616 and S637 phosphorylation of DRP1 regulates mitochondrial dynamics. In hASM cells, exposure to TNFα triggers protein unfolding and selectively activates the endoplasmic reticulum (ER) stress pathway involving the phosphorylation of inositol‐requiring enzyme 1α (pIRE1α) and subsequent splicing of X‐box binding protein 1 (XBP1s) that acts as a potent transcriptional activator for several downstream genes. We hypothesized that TNFα mediated activation of the pIRE1α/ XBP1 ER stress pathway transcriptionally activates genes that code for proteins involved in DRP1 phosphorylation at S616 leading to mitochondrial fragmentation. hASM cells were dissociated from bronchial biopsies from patients without a history of respiratory diseases and exposed to TNFα (20 ng/ ml for 6 h). Cells were also treated with Mdivi1 (50 μM for 6 h), a small‐molecule inhibitor of mitochondrial fragmentation that targets the GTPase activity of DRP1. Bioinformatic analysis revealed the presence of putative binding sites for XBP1 on the promoter region of genes that translate into kinases (cyclin dependent kinases CDK1 and CDK5, cyclin B1), which are reported to phosphorylate DRP1 at S616. Western blot and/or immunocytochemistry were employed to quantify the expression and phosphorylation status of IRE1α, DRP1, XBP1 splicing, CDKs and cyclin B1. Mitochondrial morphology was assessed by 3‐D confocal microscopy using MitoTracker green to label mitochondria. Chromatin immunoprecipitation (ChIP) and quantitative real time PCR was done to validate XBP1 target genes. Spliced XBP1 transcriptionally activates expression of CDK1, CDK5 and cyclin B1 and subsequent phosphorylation at S616 of DRP1 without significant alteration in S637 phosphorylation. As a result, TNFα induces an increase in S616/S637 phosphorylation ratio that promotes the translocation of DRP1 from cytosol to mitochondria and mediates mitochondrial fragmentation. Inhibition of DRP1 GTPase activity by Mdivi1 ameliorates DRP1 phosphorylation at S616 and significantly reduces mitochondrial fragmentation. These results reveal the mechanisms that underlie TNFα induced ER stress and downstream mitochondrial fragmentation.

Neuromuscular dysfunction increases with age and mobility restrictions decrease quality of life for the elderly. A hallmark of aging is the aggregation of proteins and damaged components within a cell, which is especially detrimental to post‐mitotic cells such as motor neurons. Autophagy, a multistep catabolic process, is responsible for the disposal of damaged cellular components. Accumulation of autophagy markers may differ across motor neuron pools consistent with differences in aging effects across muscles. Autophagy is characterized by the elongation of a double‐membrane vacuole around the tagged cargo which fuses with a lysosome and is subsequently degraded. In the present study, expression levels of factors for autophagy initiation (Beclin1), elongation (LC3I, LC3II, the LC3II/I ratio, ATG7, and the ATG5/12 complex), and degradation (p62 clearance) were determined in the cervical (CSC) and lumbar spinal cord (LSC) of male and female C57BL/6 mice at 6, 18, and 24 months of age (n=8/age) using Western blot, spanning a period where aging differences manifest in forelimb vs. hindlimb muscles. A mixed linear model with animal as a random effect was used to compare the effect of age, tissue, sex, and their interaction on the relative expression of each autophagy factor. There was an age*tissue interaction for Beclin1 at 24 mo, with greater Beclin1 expression in LSC than CSC. There was an overall effect of age on LC3I expression, with 24 mo greater than 6 mo, and no changes in LC3II expression. The LC3II/I ratio was significantly different by tissue, whereby CSC had a greater LC3II/I ratio than that of LSC. Expression of ATG7 had a tissue*sex interaction, with ATG7 in females greater in LSC than CSC. There were no changes in ATG5/12 complex. Finally, there was an age and age*tissue interaction in p62 expression. p62 was significantly greater at 24 mo than at 18 mo, and at 18mo the LSC had greater p62 expression than CSC. These results suggest greater impairment of autophagy flux in LSC compared to CSC at comparable ages, consistent with greater susceptibility to aging effects in hindlimb muscles. Future studies will evaluate motor neuron‐specific changes in markers of autophagy elongation (i.e., LC3) and p62 clearance.

Autophagy is a cellular digestion process that contributes to cellular homeostasis by the elimination of proteins and damaged organelles. Impaired autophagy has been implicated in aging‐related disorders and in neurodegenerative diseases, including motor neuron disorders. Therefore, upregulation of autophagy may serve as a promising therapeutic approach. Lanthionine ketenamine (LK), an amino acid metabolite found in mammalian brain tissue at low concentrations, activates autophagy in rat glioma and human neuroblastoma cells in vitro, as well as in hippocampus and cortex after in vivo administration. With the overall goal of using LK to induce autophagy flux to prevent age‐related motor neuron dysfunction, we recently tested the effects of two lanthionine ketenamine phosphonates (LK‐Ps) and one lanthionine ketenamine ethyl (ester) phosphonate (LKE‐P) on autophagy modulation in a mouse motor neuron‐like hybrid cell line (NSC‐34). For fluorescence visualization of autophagy flux, NSC‐34 cells were transfected with mCherry‐GFP‐LC3 plasmid. Motor neurons treated with LK analogs displayed increased autophagy flux measured by the ratio of mCherry area (lysosomes) over GFP area (autophagosomes that have not fused with lysosomes) compared to control cells. There results were corroborated by increased LC3‐II/LC3‐I ratio as measured by protein expression using Western blot; and presence of autophagic vacuoles identified using transmission electron microscopy. In conclusion, LK analogs constitute a promising tool to induce autophagy flux in motor neurons, which may serve to prevent age‐related motor axon withdrawal and muscle denervation.

Hypothermia is defined as a core body temperature of <35°C, and as body temperature is reduced the impact on physiological processes can be beneficial or detrimental. The beneficial effect of hypothermia enables circulation of cooled experimental animals to be interrupted for 1–2 h without creating harmful effects, while tolerance of circulation arrest in normothermia is between 4 and 5 min. This striking difference has attracted so many investigators, experimental as well as clinical, to this field, and this discovery was fundamental for introducing therapeutic hypothermia in modern clinical medicine in the 1950s. Together with the introduction of cardiopulmonary bypass, therapeutic hypothermia has been the cornerstone in the development of modern cardiac surgery. Therapeutic hypothermia also has an undisputed role as a protective agent in organ transplantation and as a therapeutic adjuvant for cerebral protection in neonatal encephalopathy. However, the introduction of therapeutic hypothermia for organ protection during neurosurgical procedures or as a scavenger after brain and spinal trauma has been less successful. In general, the best neuroprotection seems to be obtained by avoiding hyperthermia in injured patients. Accidental hypothermia occurs when endogenous temperature control mechanisms are incapable of maintaining core body temperature within physiologic limits and core temperature becomes dependent on ambient temperature. During hypothermia spontaneous circulation is considerably reduced and with deep and/or prolonged cooling, circulatory failure may occur, which may limit safe survival of the cooled patient. Challenges that limit safe rewarming of accidental hypothermia patients include cardiac arrhythmias, uncontrolled bleeding, and “rewarming shock.”

For the individual tongue muscles, relatively little quantification of uniaxial force, fatigue, and fiber type-specific properties has been documented. Here, we assessed uniaxial-specific force generation, fatigability, and muscle fiber type-specific properties in the superior and inferior longitudinal muscles, the transversalis, and the genioglossus in Fischer 344 rats. The longitudinal muscles produced the greatest isometric tetanic-specific forces. The genioglossus was more fatigue resistant and comprised higher proportions of I and IIa fibers.