Sieck, Gary C.

Compensatory respiratory plasticity is critical to preserve breathing capacity after phrenic denervation (DNV), when the contribution of the ipsilateral phrenic motor neuron (PhrMn) pool is abolished and the force generating capacity of the diaphragm muscle (DIAm) is compromised. In rats, ventilation is maintained after DNV, and this is most likely mediated by an increase in central drive to the contralateral PhrMn pool. The extent of central drive to PhrMns was estimated by measuring the root mean square (RMS) of DIAm EMG at 75ms (RMS 75 ) after the onset of activity. Transdiaphragmatic pressure (Pdi) was measured to estimate DIAm force. We hypothesized that after unilateral DNV central drive to the contralateral PhrMn pool increases across motor behaviors. Immediately following DNV, there was a significant increase in RMS 75 during ventilatory behaviors (eupnea, and hypoxia‐hypercapnia). RMS 75 did not change during non‐ventilatory behaviors (airway occlusion). These results indicate that after DNV there is: 1) an increase in central drive to the contralateral PhrMn pool during ventilatory behaviors such that Pdi was maintained; 2) central drive to the contralateral PhrMn pool was unchanged during more forceful (greater than ~50% Pdi max ) non‐ventilatory behaviors, suggesting that central drive was near maximum. These results highlight the compensatory plasticity in the neural control of the DIAm and the importance of measuring multiple motor behaviors.

Following spinal cord hemisection at C2 (SH), removal of descending excitatory drive to phrenic motoneurons results in ipsilateral diaphragm muscle paralysis with partial spontaneous recovery of rhythmic phrenic activity over time. Brain‐derived neurotrophic factor (BDNF) plays an important role in neuroplasticity following SH. Intraspinal transplantation of mesenchymal stem cells engineered to produce BDNF‐GFP (BDNF‐MSCs) at the time of injury promotes functional recovery of all animals, despite low levels of MSC survival at 14 days post‐SH. The goal of the present study was to determine the survival and localization of BDNF‐MSCs after intraspinal transplantation. BDNF‐MSCs were injected intraspinally at C4 (2 sites, total 2 x 10 5 cells in 4 µl) in adult male Sprague‐Dawley rats. After one and three days, the cervical spinal cord was fixed, excised, and cut longitudinally. Survival of transplanted BDNF‐MSCs was confirmed in all animals at 1 day post‐transplantation (n=5) and in five of 6 animals at 3 days post‐transplantation. BDNF‐MSCs remained localized at the site of injection in the white matter at 1 and 3 days, with an average volume of 1.1 ± 0.3 µl. There was no localization of MSCs in the vicinity of phrenic motoneurons identified by retrograde labeling with cholera toxin subunit B. Understanding the time course of stem cell survival after transplantation is important in defining windows of opportunity during which the beneficial effects of BDNF on respiratory function after spinal cord injury are executed. Supported by HL96750 and Mayo Clinic Center for Regenerative Medicine.

A transient elevation of cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ) triggers a force response in excitation‐contraction coupling. The Ca 2+ sensitivity of muscle reflects the relationship between [Ca 2+ ] cyt and force, and in skeletal and cardiac muscle is often defined as the [Ca 2+ ] cyt at which 50% force is generated (ED50). In smooth muscle, Ca 2+ sensitivity has been assessed as the ratio of peak force to peak cytosolic [Ca 2+ ] cyt . However, this conventional measurement overlooks the dynamic nature of Ca 2+ regulation of force generation. In the present study, in order to provide a dynamic index of Ca 2+ sensitivity in airway smooth muscle (ASM), we simultaneously measured [Ca 2+ ] cyt and force and determined their temporal dependence using a phase loop plot. We examined the phase loop plot assessment of Ca 2+ sensitivity in ASM under the condition in which the conditional assessment indicated an increase ( Calyculin A treatment) in Ca 2+ sensitivity . Intact ASM strips were isolated from porcine tracheas obtained from local abattoir. ASM strips were loaded with Fura‐2 AM (Ca 2+ fluorescent indicator) and stimulated using 1 mM ACh. Isometric force and [Ca 2+ ] cyt responses were simultaneously measured using a Guth Muscle Research System. ASM strips were exposed to Calyculin A (phosphatase inhibitor; 100 nM for 10 min), which was previously reported to alter static Ca 2+ sensitivity. Phase loop plots were calculated from simultaneous [Ca 2+ ] cyt and force responses. The extent of rMLC phosphorylation was determined at different time periods after ACh stimulation using Phos‐tag SDS‐PAGE (SuperSep Phos‐tag; Wako Pure Chemical Industries, Ltd.) and Western blotting. Calyculin A increased ACh‐induced isometric force in ASM strips, but it had no effect on the amplitude of evoked [Ca 2+ ] cyt transients. These results suggested that Calyculin A increased static Ca 2+ sensitivity. However, the phase loop plots showed no significant shift in dynamic Ca 2+ sensitivity with Calyculin A. These results indicate that the phase loop plot provides a valuable tool to assess dynamic Ca 2+ sensitivity of isometric force generation in ASM, which better reflects the complex process of excitation‐contraction coupling in smooth muscle. These results further indicate that dynamic Ca 2+ sensitivity of force generation in ASM is independent of rMLC phosphorylation. Support or Funding Information Supported by NIH grant HL126451 (GCS) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Age related loss of larger phrenic motor neurons (PhMNs) may underlie diaphragm muscle sarcopenia. We found increased mitochondrial fragmentation in larger PhMNs, similar to that reported to be associated with motor neuron loss in amyotrophic lateral sclerosis (ALS). In rodents, we observed diminished brain‐derived neurotrophic factor (BDNF) signaling via its high affinity TrkB.FL receptor in older animals. TrkB.FL is expressed on the outer mitochondrial membrane, and in ALS models, BDNF/TrkB.FL signaling promotes motor neuron survival and mitochondrial fusion. In the present study, we hypothesized that enhancing BDNF/TrkB.FL signaling increases Mfn2 expression, thereby promoting mitochondrial fusion. Studies were conducted using a mouse motor neuron‐like cell line (NSC‐34), which were differentiated by serum depletion until neurite structures were observed. The differentiated NSC‐34 cells were treated with BDNF (12.5 ng/ml for 1 h) and then loaded with MitoTracker Green to image mitochondria using a Nikon A1R confocal microscope (60× oil immersion 1.4 NA). BDNF treatment increased both form factor and aspect ratio of mitochondria by ~ 20% and 15%, respectively, while also causing a threefold increase in expression of Mfn2, indicating mitochondrial fusion. These experiments were performed in six different trials with two replicates each. The observed relationship between BDNF/TrkB.FL signaling and mitochondrial fusion/fragmentation proteins supports our hypothesis. These results are consistent with diminished BDNF/TrkB.FL signaling underlying mitochondrial fragmentation and dysfunction in age‐related PhMN loss. Support or Funding Information Supported by NIH grant AG44615 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Ca 2+ sensitivity plays a critical role in regulating cardiac contraction, and pathophysiological changes in Ca 2+ sensitivity of cardiac contraction may significantly impact cardiac function. For example, oxidative stress causes myocardial dysfunction, which is due in part to a decrease in Ca 2+ sensitivity. Oxidative stress (e.g., elevated H 2 O 2 ) underlies, at least in part, various conditions of cardiac dysfunction including ischemia‐reperfusion injury, heart failure, hypertension, and atherosclerosis. In cardiac muscle, Ca 2+ sensitivity can be dynamically assessed using a phase loop plot of concurrently measured cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ) and sarcomere length shortening (Spurgeon et al J Physiol 447:83–102, 1992; Han et al Am J Physiol 298:890–897, 2010; Schaible et al Am J Physiol 310:1533–40, 2016). We hypothesized that oxidative stress reversibly reduces Ca 2+ sensitivity of cardiomyocytes within minutes. Cardiomyocytes were isolated from adult rat hearts (Sprague Dawley) using a modified Langendorff system. Cardiomyocytes were loaded with Fura‐2 AM (Ca 2+ fluorescent indicator; 1 mM) and electrically stimulated every 2 s. [Ca 2+ ] cyt and sarcomere shortening responses to stimulation were simultaneously measured using an IonOptix System. After stabilization for 30 min at 35°C, cardiomyocytes were exposed to PSS for 10 min, then to H 2 O 2 (200 μM) for 10 min and finally to either PSS washout or DTT (1 mM)‐induced reduction for 10 min. H 2 O 2 ‐induced oxidative stress increased basal [Ca 2+ ] cyt and the amplitude of the evoked [Ca 2+ ] cyt response. H 2 O 2 ‐induced oxidative stress also reduced the extent of sarcomere shortening and slowed the kinetics of shortening/relaxation. As a result, H 2 O 2 ‐induced oxidative stress reduced Ca 2+ sensitivity reflected by a rightward shift in the phase loop plots. Interestingly, with both PSS washout and DTT‐induced reduction Ca 2+ sensitivity was further reduced. These results support the hypothesis that H 2 O 2 ‐induced oxidative stress reduces Ca 2+ sensitivity of cardiomyocytes, but this effect was not immediately reversed, but indeed worsened by PSS washout or by DTT‐induced reduction. Together, these results provide insight into the dynamic effects of oxidative stress on Ca 2+ sensitivity of cardiomyocytes that may be reflected under pathological conditions. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Lipid rafts are structurally unique components of plasma membranes, crucial for neural development and function. The uptake and transport of lipid raft associated molecules in motor neurons is poorly characterized under normal and pathologic conditions, including advanced aging effects. Lipid rafts are particularly important for motor neurons as they shuttle cargo between peripheral and central compartments. The cholera toxin subunit B (CTB) binds the cell‐surface ganglioside GM1, which is internalized and transported via lipid rafts in motor neurons, and thus is commonly used to investigate lipid rafts. We hypothesized firstly, that axon terminal cargo loading into lipid rafts will be different across fiber types and secondly, that uptake at axon terminals is not affected by activity at the neuromuscular junction (NMJ). Ex vivo diaphragm muscle and phrenic nerve preparations from 6‐month old female and male Fischer 344 rats were harvested. Morphological analyses of pre‐ and post‐synaptic structures at the NMJ were conducted using confocal imaging of fluorescently labeled anti‐synaptophysin and alpha‐bungarotoxin, respectively. NMJs were identified at type I or IIa vs. type IIx and/or IIb fibers using validated morphological characteristics. Quantitative analyses of CTB fluorescence at each type‐identified NMJ were conducted. CTB fluorescence was identified in all NMJs, and there were no fiber type differences in CTB uptake. Phrenic nerve stimulation had no effect on any parameters of CTB uptake, consistent with the exclusive involvement of lipid rafts rather than synaptic vesicle membrane uptake. Taken together, we conclude that lipid rafts are an important constituent of axon terminal membranes, that there are no differences in lipid raft uptake across motor unit types, and that lipid raft uptake is not affected by motor unit activity. 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 population at very old age is increasing dramatically throughout the world. Aging adults experience increased health and medical needs associated with debility and sarcopenia (i.e., muscle atrophy and force loss). Respiratory complications and diseases are common causes of morbidity and mortality in the elderly. Sarcopenia of respiratory muscles such as the diaphragm muscle (DIAm) may contribute to respiratory complications since the DIAm, the main inspiratory muscle in mammals, plays an important role in ventilation and generating the forces necessary to maintain airway clearance. Presently, there is limited information about aging effects into very old age. Specifically, whether DIAm sarcopenia is exacerbated in old age and if it will effect ventilation into very old age are presently unknown. We hypothesized that DIAm sarcopenia in very old mice (>30 mo; ~30% survival) will result in reduced DIAm force generation across motor behaviors, exhibit impaired ventilation, decreased isometric force, and reduced fiber cross‐sectional area compared to old (24 mo; ~80% survival), and young (6 mo, 100% survival). C57BL/6 × 129 female and male mice (n = 30) at 6 mo, 24 mo, and >30 mo were used to examine transdiaphragmatic pressure (Pdi) generation during eupnea, exposure to hypoxia‐hypercapnia, as well as higher force behaviors in airway occlusion, and/or bilateral phrenic stimulation. Ventilation was assessed using whole body plethysmography and venous blood gases. Isometric force was examined via physiological muscle force testing, and fiber cross‐sectional area was determined in immunohistochemically labeled sections across fiber types. There was a significant reduction in Pdi between young and old mice for higher force behaviors – tracheal occlusion and bilateral phrenic nerve stimulation, (p < 0.001); however, there was no further decline in Pdi for all behaviors between old and very old mice. There was no evidence of impaired ventilation (ventilatory parameters – respiratory rate, minute ventilation, or tidal volume) across age groups, and no evidence of differences between sex in any of the measurements. A loss of ability to generate higher levels of force necessary to maintain airway clearance may predispose older individuals to recurrent infection. Understanding the mechanisms of sarcopenia in old age will be important in mitigating the effects of aging on quality of life and maintaining the health span. Support or Funding Information This work was supported by grants R25‐GM075148, R01‐AG044615, and R01‐AG057052 from the National Institutes of Health. 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 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 .

Intracellular Ca 2+ regulation in human airway smooth muscle (hASM) involves plasma membrane (PM) Ca 2+ influx, including that triggered by sarcoplasmic reticulum (SR) Ca 2+ depletion, i.e. store‐operated Ca 2+ entry (SOCE). While transient receptor potential channels (e.g. TRPC3, TRPC4) are thought to mediate SOCE, the mechanisms triggering or regulating SOCE are being actively investigated. Recent studies suggest that stromal interaction molecule 1 (STIM1) acts as a signaling mediator between PM and SR, potentially regulating Ca 2+ entry via SOCE. In hASM, fluorescence microscopy and western blot analyses were used to verify the presence of STIM1. SR Ca 2+ depletion with 10μM cyclopiazonic acid (CPA) resulted in increased STIM1 expression in the PM fraction, compared to controls. Transfection with 50nM STIM1 siRNA for 48h significantly decreased STIM1 levels. In cells where [Ca 2+ ] i was measured using fura 2‐AM, SOCE was significantly reduced in cells transfected with STIM1 siRNA. Green‐fluorescent protein (GFP)‐tagged STIM1 transfection showed intracellular localization in non‐stimulated cells, with increased PM staining following CPA exposure. Subsequent immunostaining revealed co‐localization of STIM1 with TRPC3 and TRPC4. These results indicate that STIM1 is present in hASM and suggest a role for this protein in regulating SOCE. This work has been supported by NIH grants UL1RR024150‐01(CR to CMP, CR20 and Early Career Development Award to YSP), HL74309 (GCS), and the Department of Anesthesiology (CMP, YSP), Mayo Clinic College of Medicine, Rochester, MN