Gary C. Sieck, Ph.D. - US grants

DESCRIPTION (Adapted from the applicant's abstract): This investigation will evaluate the underlying relationships between changes in myosin/troponin expression during development and specific morphological and functional characteristics of muscle fibers. In previous studies, the investigators found dramatic changes in myosin heavy chain isoforms, morphology and function in diaphragm fibers during development. This study will explore the relationships between these variables. Specific aim 1 will determine the underlying basis for the postnatal establishment of morphological differences among diaphragm fibers. The hypothesis to be tested is that fiber type differences in postnatal growth reflect differences in synthesis rate of myosin heavy chain (MHC) isoforms that result in a higher MHC content of fibers expressing MHC2x and MHC2b isoforms compared to fibers expressing MHCslow and MHC2A isoforms. Specific aim 2 will determine the underlying basis for the postnatal establishment of fiber type differences in maximum specific force (Po). The hypothesis to be tested is that postnatal increases in Po and adult fiber type difference in Po primarily reflect the influence of varying MHC content. Specific Aim 3 will determine the underlying basis for the postnatal establishment of fiber type differences in Ca+2 sensitivity. The hypothesis to be tested is that fiber type differences in the dependence of force on myoplasmic Ca+2 relate to the expression of different troponin C isoforms; however within a single fiber, Ca+2 sensitivity is modulated by the extent of myosin light chain phosphorylation. Specific Aim 4 will determine the underlying basis for the postnatal establishment of fiber type differences in actomyosin ATPase activity. The hypothesis to be tested is that fiber type differences in actomyosin ATPase activity reflect the combined influence of MHC content, the fraction of cross bridges in the force generating state (alpha-fs) and the apparent rate constant for cross bridge detachment (g-app). With shortening contractions, the increase in actomyosin ATPase activity varies across fiber types, reflecting differences in the impact of active shortening on alpha-fs and g-app.

We are requesting support for an international symposium on "Respiratory Muscles and Their Neuromotor Control" to be held at the UCLA Conference Center during July 1986. The program of this symposium has been officially ratified as a satellite meeting of the triennial congress of the International Union of Physiological Sciences to be held in Vancouver, British Columbia. This Symposium has three major aims: 1) To bring together researchers who are using various approaches to study the neural control of breathing muscles. 2) To critically review recent advances in the major areas of respiratory neuromotor control. 3) To define important areas for future research and to encourage increased collaboration between the diverse research approaches used to study respiratory muscles and their neuromotor control. No previous symposium has focused on the neuromotor control of breathing muscles. An understanding of this neural control is important since many diseases can affect the control of ventilatory muscles at various levels. Therefore, the proposed symposium is timely and should provide an important forum for basic researchers and clinicians to interact and share information.

The proposed studies will examine the influence of altered activation of the diaphragm of the fatigue resistance and oxidative capacity of muscle units. Prolonged diaphragm disuse will be imposed by blocking neural traffic in the right phrenic nerve using tetrodoxtoxin (TTX). In both control and TTX treated animals, diaphragm motor units will be isolated by microdissection of C5 ventral root filaments. Diaphragm motor units will be classified into different types, based on standard criteria commonly used in the study of hindlimb motor units, e.g. fast- and slow-twitch motor units will be distinguished using the "sag" test and a 2-min fatigue test will be used to separate units into fatigable, intermediate and fatigue resistant types. Muscle fibers will be classified as Type I, IIA or IIB based on differences in staining for myosin ATPase at different preincubation pH's. The oxidation capacity of muscle fibers will be determined by quantifying the histochemical reaction for succinate dehydrogenase (SDH) using a microphotometeric technique. The cross-sectional areas of muscle fibers will also be quantified. In population studies, the influence of prolonged disuse of the diaphragm on the distribution of: 1) Motor unit types; 2) Muscle fiber types; 3) Fiber SDH activities; and 4) Fiber cross-sectional areas will be determined. In another series of studies, muscle fibers belonging to single motor units in both control and TTX treated diaphragms will be identified using the method of gylcogen depletion. The variability of SDH activities among muscle unit fibers will be compared to that for non-unit fibers. The relationship between motor unit fatigue resistance and the mean SDH activity of muscle unit fibers will be determined. In these studies, we will control for the potential influence of precontractile failure of some muscle fibers by comparing the properties of motor units that show a decrease in motor unit action potential (MUAP) amplitude during the fatigue test with those of units showing no change in MUAP amplitude. The results of these studies will be clinically important for several reasons. First, the long-term maintenance of patients on mechanical ventilators may seriously impair the contractile and fatigue properties of the diaphragm. Secondly, detremental adaptive responses of the diaphragm to altered levels of activation may contribute to the pathophysiology of certain respiratory diseases. Finally, understanding how the diaphragm adapts to altered levels of activation may provide the basis for designing treatments effective in improving ventilatory muscle strength and endurance.

The overall goal of the proposed studies is to gain insight into how phrenic motoneurons and diaphragm muscle (DIAm) fibers interact in the remodeling (plasticity) associated with conditions of altered use. Specifically, we will examine the role of phrenic motoneuron activity and the expression of specific neurotrophic factors (NT3 and NT4/5) and their receptors (TrkC and TrkB) in plasticity at DIAm fibers and neuromuscular functions (NMJs) under three experimental conditions of DIAm inactivity: 1) Unilateral denervation (DNV), where communication between phrenic motoneurons and DIAm fibers is complete disrupted, and motoneuron activity increases; 2) Unilateral tetrodotoxin (TTX) nerve blockade, where communication between phrenic motoneurons and DIAm fibers remains intact, and motoneuron activity increases; and 3) Spinal hemisection (SH) at C2, where communication between phrenic motoneurons and DIAm fibers remains intact, and motoneurons are inactive. In previous studies, we demonstrated that DNV and TTX are associated with a decrease in specific force of DIAm strips, a selective atrophy of type IIx and IIb DIAm fibers, and a decrease in the relative expression of MHC/2X and MHC/2B isoforms, w2hereas SH induced little change in DIAm fiber structure and function. In contrast, we found that in the SH DIAm, there is an expansion of NMJs innervating type IIx and IIb fibers. The proposed studies will extend these previous observations by evaluating the fiber type dependence of these adaptations and the role of selected neurotrophic factors. Specific factors: 1) To determine the influence of DNV and TTX on a) specific force, and 2) MHC content per half sarcomere of single DIAm fibers. 3) To determine the influence of DNV, TTX and SH on neurotrophic factor and receptor expression in a) DIAm, b) phrenic motoneurons, and c) NMJs. 4) To determine the effect of neurotrophic facto treatment on a) mechanical properties & MHC content of single DIAm fibers, and b) MHC expression and fractional synthesis rate of MHC isoforms in the DIAm. 5) To determine the effect of neurotrophic facto treatment on NMJ morphology and neuromuscular transmission.

DESCRIPTION: (Adapted from the abstract) The long-term goals of the proposed research are to understand the effects of volatile anesthetics on: 1) the regulation of [Ca2+]i; 2) the coupling between elevated [Ca2+]i and mechanical responses (excitation- contraction); and 3) the Ca2+ sensitivity of force generation in ASM cells. The proposed studies will use real-time confocal microscopy to examine the effects of halothane and sevoflurane on acetylcholine- induced [Ca2+]i oscillations in localized regions within freshly dissociated porcine ASM cells. Flash photolytic release of caged Ca2+ and ATP will be used to determine effects of volatile anesthetics on the intracellular process contributing to the delays during excitation- contraction coupling. There are four major specific aims in the proposed studies: 1) To determine the effect of volatile anesthetics on acetylcholine-induced [Ca2+]i oscillations, 2) To determine whether volatile anesthetics affect second messenger (IP3, and cADPR) production in response to acetylcholine, 3) To determine whether volatile anesthetics affect IP3 and cADPR-mediated SR Ca2+ release, and 4) To determine the effects of volatile anesthetic on the Ca2+ sensitivity of force generation and the dynamic coupling between elevated [Ca2+]i and ASM contraction. With regard to [Ca2+]i regulation, the authors hypothesize that volatile anesthetics 1) increase IP3 production but decrease cADPR production; 2) decrease SR Ca2+ content via an IP3- induced "leak" and reduced SR Ca2+ reuptake, thereby decreasing the peak amplitude of [Ca2+]i oscillations; and 3) lower the sensitivity of Ca2+- induced Ca2+ release (CICR) via decreased cADPR production, thereby slowing [Ca2+]i oscillation frequency and propagation velocity. With regard to the coupling between elevated [Ca2+]i and ASM contraction, the investigators hypothesize that volatile anesthetics 1) decrease the steady-state Ca2+ sensitivity of force generation during muscarinic stimulation and 2) delay excitation-contraction coupling.

DESCRIPTION (provided by applicant): Volatile anesthetics such as halothane, isoflurane and sevoflurane are potent bronchodilators. The long-term goal of the proposed research is to understand the mechanisms by which volatile anesthetics relax airway smooth muscle (ASM). During the previous funding period, we found that anesthetics inhibit ACh-induced intracelllar calcium ([Ca2+]i) oscillations. Paradoxically, anesthetics enhance both baseline and ACh-induced IP3 production. Accordingly, the first major hypothesis of the present proposal is that volatile anesthetics reduce agonist-induced [Ca2+]i responses in ASM by depleting sarcoplasmic reticulum (SR) Ca2+ stores. In ASM, agonist-induced elevation of [Ca2+]i involves Ca2+ influx and SR Ca2+ release via IP3 and ryanodine receptor (RyR) channels. SR Ca2+ release through RyR channels is mediated via Ca2+ itself as well as the second messenger, cyclic ADP ribose (cADPR). SR Ca 2+ stores are maintained by active Ca2+ reuptake and by plasma membrane Ca2+ influx triggered by SR depletion (Ca2+ release-activated Ca2+ influx; Icrac). In the proposed studies, we will examine volatile anesthetic effects on these specific mechanisms of SR Ca2+ regulation. In addition to affecting [Ca2+]i regulation, volatile anesthetics may also decrease ASM contractility by interfering with mechanisms distal to Ca2+. Accordingly, our second major hypothesis is that volatile anesthetics decrease ASM contractility by interfering with EC coupling. Specific Aims: 1) To determine the mechanisms by which volatile anesthetics affect regulation of IP3 and cADPR levels in ASM; 2) To determine the mechanisms by which volatile anesthetics affect SR Ca2+ release in ASM; 3) To determine the mechanisms by which volatile anesthetics affect SR Ca2+ reuptake in ASM; 4) To determine the effect of volatile anesthetics on Ca2+release activated Ca2+influx (Icrac) in ASM; and 5) To determine the effect of volatile anesthetics on EC coupling in ASM. With reference to [Ca2+]i, we specifically hypothesize that anesthetics deplete SR Ca2+ stores by: increasing IP3 levels, increasing SR Ca 2+ "leak" through both IP3 and RyR channels, decreasing SR Ca2+ reuptake and decreasing Ca 2+ influx via Icrac. Furthermore, we hypothesize that anesthetics inhibit EC coupling by interfering with Ca2+-calmodulin interactions and myosin light chain activation. The proposed studies will use real-time confocal microscopy of [Ca2+]i and biochemical techniques to examine the effects of volatile anesthetics on these mechanisms of [Ca2+]i and force regulation in freshly dissociated porcine ASM ceils. These interactions will be examined in the context of unstimulated (baseline) conditions as well as with ACh stimulation. The studies will initially focus on the effects of halothane; however, when significant effects are observed, the effects of isoflurane and sevoflurane will also be explored.

[unreadable] DESCRIPTION (provided by applicant): During postnatal development, DIAm fibers display dramatic growth that is disproportionate across fibers expressing different myosin heavy chain (MHC) isoforms. Postnatal increase in specific force of DIAm fibers also varies across fibers expressing different MHC isoforms. Unilateral denervation (DNV) dramatically retards postnatal growth of DIAm fibers and reduces specific force. These data suggest that nerve-derived factors (such as the family of neurotrophins) play a key role in postnatal muscle fiber growth. We hypothesize that neurotrophins modulate the postnatal growth of DIAm fibers and MHC protein expression and thereby affect specific force. The major goals of the proposed studies are: 1) to explore the mechanisms regulating MHC protein expression during postnatal growth of DIAm fibers, 2) to explore the effects of TrkB receptor activation on MHC protein expression, 3) to explore the effects of DNV (i.e., removal of neurotrophin influence) on MHC protein expression, and 4) to examine the relationship between postnatal changes in MHC protein content and changes in specific force generated by DIAm fibers. There are four specific aims in the proposed studies: Specific Aim #1: To examine the impact of DNV on postnatal changes in myonuclear domain size in DIAm fibers Hypothesis la - Following DNV, myonuclear domain size decreases and this effect varies with postnatal age and across different MHC isoforms. Hypothesis lb - In cultured myotubes/myofibers, TrkB receptor activation increases myonuclear domain size. Specific Aim #2: To evaluate the impact of DNV on postnatal changes in MHC isoform transcription in DIAm fibers. Hypothesis 2a - Following DNV, MHC transcription decreases and this effect varies with postnatal age and across different MHC isoforms. Hypothesis 2b - In cultured myotubes/myofibers, TrkB receptor activation increases MHC transcription. Specific Aim #3: To evaluate the impact of DNV on postnatal changes in MHC protein content per haft sarcomere and MHC protein turnover in DIAm fibers. Hypothesis 3a - Following DNV, MHC protein content per half sarcomere decreases and this effect varies with postnatal age and across different MHC isoforms. Hypothesis 3b - Following DNV, the fractional synthesis rate of MHC decreases while degradation rate increases, and these effects on MHC turnover vary with postnatal age and across different MHC isoforms. Hypothesis 3c - In cultured myotubes/myofibers, TrkB receptor activation increases MHC protein expression. Specific Aim #4: To evaluate the impact of DNV on postnatal changes in specific force of DIAm fibers. Hypothesis 4a - Following DNV, specific force of DIAm fibers decreases and this effect varies with postnatal age and across fibers expressing different MHC isoforms. Hypothesis 4b - Following DNV, the decrease in DIAm fiber specific force reflects both a decrease in MHC content per half sarcomere and a decrease in the force per cross bridge. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Airway hyperreactivity in diseases such as asthma involves enhanced airway smooth muscle (ASM) contraction due either to increased intracellular Ca2+ ([Ca2+]i) and/or increased Ca2+ sensitivity (force for a given [Ca2+]i). Airway inflammation is a key aspect of airways disease, and exposure to several inflammatory mediators (such as tumor necrosis factor (TNFa) and interleukin-13 (IL-13)) increase ASM contractility. Several studies showed that cytokines increase agonist-induced [Ca2+]i responses in ASM, thus linking In the current proposal, we will explore inflammation-induced changes in [Ca2+]i regulatory mechanisms that result in overall increased [Ca2+]i responses. In ASM, Ca2+ influx in response to SR depletion (store-operated Ca2+ entry;SOCE) is enhanced by cytokines. Preliminary data suggests that Na+/Ca2+ exchange (NCX)-mediated influx is enhanced by cytokines. Such enhancement of SOCE and influx-mode NCX would lead to increased [Ca2+]i levels. Reduction in [Ca2+]i is normally achieved by plasma membrane (PM) efflux mechanisms (perhaps including NCX-mediated efflux), and by SR Ca2+ reuptake via SR ATPase (SERCA). Organelles such as mitochondria can buffer Ca2+ and alter Ca2+ availability for SR refilling. Normally, these mechanisms help maintain basal [Ca2+]i at low levels, while SR Ca2+ stores are replete until agonist stimulation when [Ca2+]i rises as SR stores deplete. Preliminary studies suggest that SERCA and mitochondrial Ca2+ buffering are impaired by inflammation. Based on these contrasting preliminary findings of enhanced Ca2+ influx, but decreased sequestration or efflux, our central hypothesis is that inflammation promotes mechanisms that increase [Ca2+]i, but impairs those that decrease [Ca2+]i. This leads to an overall increase in basal [Ca2+]i as well as enhanced [Ca2+]i responses to agonist stimulation. In this regard, we propose that PM vs. intracellular mechanisms functionally interact in [Ca2+]i homeostasis under normal circumstances, and that disruption of these mechanisms and their interactions with inflammation leads to increased [Ca2+]i. Our overall approach will be to use human ASM cells or tissue strips to examine the above mechanisms with or without exposure to pro-inflammatory cytokines (TNFa, IL-13). Studies will use complementary techniques including molecular biology (siRNA;overexpression), imaging of [Ca2+]i, [Na+]i and luminal (SR) Ca2+, real-time confocal imaging of fluorescently- tagged proteins, as well as force measurements to address these aims. Our Specific Aims are: decrease [Ca2+]i Aim 1: To determine the influence of inflammatory cytokines on STIM1, STIM2 and Orai1 interactions in human ASM regulation;Aim 2: To determine the influence of inflammatory cytokines on NCX and its role in [Ca2+]i regulation in human ASM;Aim 3: To determine the influence of inflammatory cytokines on mitochondria and its role in [Ca2+]i regulation in human ASM;Aim 4: To determine the influence of inflammatory cytokines on SERCA and its role in [Ca2+]i regulation in human ASM;[Ca2+]i Aim 5: To determine the influence of inflammatory cytokines on the overall contribution of [Ca2+]i regulatory mechanisms to contractility in human ASM. PUBLIC HEALTH RELEVANCE: There is increasing recognition that abnormalities in airway smooth muscle contractility contribute to exaggerated airway narrowing and accompanying shortness of breath in clinically-important diseases such as asthma and chronic bronchitis. Airway contractility is highly dependent on intracellular calcium levels. The proposed studies will examine the mechanisms by which intracellular calcium is controlled in human airway smooth muscle. These studies will the foundation for better understanding of airway diseases, and potential development of new therapeutic targets.

DESCRIPTION (provided by applicant): The proposed studies address very basic questions regarding plasticity and recovery of respiratory function following upper cervical spinal cord injury (SCI). There are about 11,000 new cases of SCI in the United States each year, with nearly 500,000 people affected. Most SCI's are incomplete with some sparing of spinal cord pathways. Among SCI patients, about 52% involve the cervical spinal cord and in many cases this results in impairment of rhythmic phrenic nerve activity and paralysis of the diaphragm muscle. Some of these SCI patients must be maintained on long-term mechanical ventilation, with associated higher morbidity and mortality rates. Clearly, it is important to understand how rhythmic phrenic activity can be restored in these SCI patients and this is a key objective of the proposed research. It is well established that excitatory premotor drive to phrenic motoneurons emanates predominantly from the ipsilateral medulla. As a result, after C2 spinal cord hemisection (SH) ipsilateral excitatory input is removed and rhythmic phrenic activity disappears on the affected side. However, there is a latent contralateral excitatory premotor input to phrenic motoneurons that can be strengthened with time after SH (neuroplasticity) leading to functional recovery of rhythmic phrenic activity. Converging evidence suggests that neurotrophins (e.g., brain- derived neurotrophic factor - BDNF) acting through tropomyosin related kinase receptors (e.g., TrkB) play an important role in neuroplasticity. Our central hypothesis is that functional recovery of rhythmic phrenic activity after SH is enhanced by an increase in TrkB.FL signaling in phrenic motoneurons. Our long-term goal is to develop an effective therapy to increase TrkB.FL expression in phrenic motoneurons and thereby promote functional recovery after upper cervical SCI. We propose the following five specific aims: 1) To examine the impact of reduced TrkB receptor expression and/or signaling in phrenic motoneurons on functional recovery of rhythmic phrenic activity after SH;2) To determine whether the continuing presence of neurotrophins (long-term effect) increases the relative expression of TrkB.FL in phrenic motoneurons after SH;3) To determine changes in downstream pathways of TrkB.FL signaling in phrenic motoneurons after SH;4) To determine whether time-dependent changes in TrkB signaling in phrenic motoneurons post-SH mediate the acute enhancing effect of intrathecal BDNF treatment on functional recovery during different behavioral conditions;and, 5) To determine whether functional recovery of rhythmic phrenic activity after SH is enhanced by increasing TrkB.FL expression in phrenic motoneurons using intrapleurally-administered gene transfer therapy. PUBLIC HEALTH RELEVANCE: Spinal cord injury is a devastating problem that affects about 500,000 people in the United States, with 11,000 new cases each year. The diaphragm muscle is the most important inspiratory muscle and it is paralyzed or seriously impaired in many cases of spinal cord injury. The proposed studies will provide important new information regarding the mechanisms underlying recovery of phrenic nerve activity and diaphragm function following spinal cord injury.

The Department of Physiology & Biomedical Engineering (BME) at Mayo Clinic has a long and rich history of preparing pre- and postdoctoral students for academic careers in a biomedical research environment that is increasingly more technological and complex. We strongly believe that a training grant that takes the novel approach of encouraging and nurturing biomedical research skills alongside computational, mathematical and engineering skills will create a unique cadre of future leaders in biomedical research related to lung disease. Under the auspices of the Training Program in Lung Physiology and Biomedical Engineering we successfully implemented a multifaceted program to train the next generation of biomedical researchers in lung physiology and disease. We recruited highly competitive predoctoral and postdoctoral trainees from different backgrounds (including clinicians (MDs)) who highlighted the success of our multidisciplinary approach. Accordingly, in this first renewal of our T32, the primary objectives of the training program will continue to be to train three groups of trainees for biomedical research careers in lung physiology and disease: 1) Predoctoral PhD (or MD/PhD) students with undergraduate backgrounds in engineering, mathematics or physics; 2) Postdoctoral PhD scientists with backgrounds in engineering, mathematics, physics or basic biomedical sciences; and 3) Postdoctoral MD or MD/PhD clinician-scientists. To achieve our objectives, we are requesting support for 2 predoctoral students (PhD or MD/PhD) and 6 postdoctoral trainees (with PhD and/or MD). From a pool of highly competitive eligible candidates with diverse backgrounds, we plan to recruit: 1) Predoctoral students via the Mayo Graduate (PhD students) and Medical (MD/PhD students) Schools; 2) Postdoctoral PhD scientists from applicants working in or applying to faculty laboratories; and 3) Postdoctoral MD or MD/PhD clinician-scientists from the large pool of residents or fellows from participating clinical departments (especially Anesthesiology, Pulmonary/Critical Care, Radiology and Surgery). A total of 20 highly-funded faculty mentors were selected based on their outstanding pre- and postdoctoral training records and their abilities to support trainees via extramural funding. The program is jointly led by Drs. Gary Sieck, PhD (Prof of Physiology, BME, and Anesthesiology; contact PI) and Y.S. Prakash, MD, PhD (Professor and Chair of Physiology and BME, Vice-Chair, Anesthesiology; Co-PI) who have extensive experience in biomedical research and demonstrated leadership in graduate and postgraduate education as well as administration at departmental, institutional and national levels. Individual trainee needs are met by common formal didactic program in lung physiology and BME, customized elective coursework, and training in writing manuscripts, grant applications, presentations, professional networking and interview skills. Success of the training program will be determined by retention and placement of trainees in academia at all levels of career development and ultimately as established, extramurally-funded biomedical researchers.

By 2030, ~70 million people in the USA will be >65 years of age and ~10 million will be >85 years old. With aging of our population, there will be an increased incidence of age-related muscle wasting and weakness (sarcopenia), which is a significant predictor of chronic disease and mortality in the elderly. Previously, we found that sarcopenia affects the diaphragm muscle (DIAm) with atrophy of more fatigable type IIx and/or IIb muscle fibers, and a reduction in maximum specific force, making the DIAm considerably weaker in old age. Important to the proposed studies, we found that in older rats there are fewer large phrenic motor neurons (PMNs) that comprise more fatigable motor units. This raises the intriguing possibility that DIAm sarcopenia results from the loss of larger PMNs, leading to denervation of type IIx and/or IIb muscle fibers and DIAm weakness. In support, we found that DIAm sarcopenia is associated with impaired performance of higher force airway clearance behaviors (e.g., coughing, sneezing), which may underlie the increased risk of airway infections in older adults. Premise: The underlying cause of age-related PMN loss is unclear. Certainly with breathing, smaller PMNs are recruited more frequently, and thus their energy demands are higher. Accordingly, it is not surprising that in preliminary studies, we found that mitochondria in smaller PMNs are more fused and have a higher volume density. In diseases such as amyotrophic lateral sclerosis (ALS), mitochondrial fragmentation appears to precede the loss of motor neurons, which is associated with decreased Mfn2 and increased Drp1 expression. Indeed, experimental interventions that support mitochondrial fusion or inhibit fission appear to ameliorate motor neuron dysfunction and degeneration in ALS models. It is also important to note that brain-derived neurotropic factor (BDNF) signaling through its high affinity receptor (TrkB.Fl) promotes motor neuron survival, and mitochondrial fusion, suggesting there may be a link. In support, TrkB.Fl is co-localized to the mitochondrial outer membrane, and in preliminary studies we found that enhanced BDNF/TrkB signaling promotes mitochondrial fusion in NSC34 cultured motor neurons. Conceptual Framework: We hypothesize that the age-related loss of larger PMNs is related to mitochondrial fragmentation, which results from decreased Mfn2 and increased Drp1 expression, and is mitigated by enhanced BDNF/TrkB signaling. Specific Aim 1: To test the hypothesis that size-dependent differences in mitochondrial morphology in PMNs are exacerbated with aging and relate to differences in Mfn2 and Drp1 expression. Specific Aim 2: To test the hypothesis that the age-related loss of larger PMNs is related to mitochondrial fragmentation, which results from decreased Mfn2 and increased Drp1 expression. Specific Aim 3: To test the hypothesis that enhanced BDNF/TrkB.Fl signaling in PMNs mitigates age-related PMN loss, through an effect on Mfn2 and Drp1 expression and mitochondrial morphology.

? DESCRIPTION (provided by applicant): The proposed studies will address fundamental questions regarding the mechanisms underlying inflammation-induced enhancement of both hyperactive (contractile) and proliferative (synthetic) states of human airway smooth muscle (hASM), which are hallmarks of asthma. Identifying these mechanisms is the key to developing novel therapeutic targets for asthma. Our central hypothesis is that in hASM, inflammatory cytokines induce sarco-endoplasmic reticulum (SR/ER) stress leading to reduced expression of the mitochondrial fusion protein mitofusin 2 (Mfn2), and that this pathway plays a central role in asthma by triggering both hyper reactive (contractile) and proliferative (synthetic) states. Four Specific Aims are proposed: Specific Aim 1: To determine the impact of inflammatory cytokines on SR/ER stress, Mfn2 expression and mitochondrial fragmentation. In this aim, we will use dissociated hASM cells and tissue from normal and asthmatic patients to test the hypothesis that inflammatory cytokines trigger SR/ER stress due at least in part to an increase in ROS generation, and that SR/ER stress leads to reduced Mfn2 expression and increased mitochondrial fragmentation. Specific Aim 2: To determine the functional impact of SR/ER stress and reduced Mfn2 expression. In this aim, we will use dissociated hASM cells and tissue from normal and asthmatic patients to test the hypothesis that in hASM, inflammatory cytokine-induced SR/ER stress and reduced Mfn2 expression uncouples mitochondria and the SR/ER, thereby reducing mitochondrial Ca2+ buffering leading to elevated [Ca2+]cyt and force responses to agonist stimulation. Specific Aim 3: To determine the impact of SR/ER stress and reduced Mfn2 on hASM cell proliferation. In this aim, we will use dissociated hASM cells and tissue from normal and asthmatic patients to test the hypothesis that inflammatory cytokines increase hASM cell proliferation as a result of decreased Mfn2 expression. Specific Aim 4: To determine the efficacy of therapeutic approaches targeting SR/ER stress in alleviating airway hyper reactivity and remodeling in a mouse model of asthma. In this aim, we will use a mixed allergen mouse model to test the hypothesis that targeting SR/ER stress using chemical chaperones (e.g., 4-PBA, TUDCA) will provide an effective therapeutic strategy to reverse airway hyperactivity and blunt remodeling associated with asthma.

ABSTRACT The goal of this application is to determine the role of trophic interactions in the susceptibility (or resilience) to the effects of aging on the neuromuscular system. Age-related neuromuscular dysfunction is an important determinant of overall health, limiting independence, increasing frailty and predisposing individuals to age- related co-morbidities and mortality. Interactions between motoneurons and the muscle fibers they innervate determine muscle fiber properties and have a significant impact on muscle function throughout the lifespan. Motoneuron-muscle fiber interactions are likely exerted via trophic factors that vary across muscle groups. Brain-derived neurotrophic factor (BDNF) acting via its high-affinity receptor tropomyosin related kinase receptor (TrkB) has a known role in the maintenance of the adult NMJ. However, the role of BDNF/TrkB signaling in old age is not presently understood. Exciting recent studies show that inhibition of TrkB kinase activity exerts deleterious effects on neuromuscular transmission that vary across age groups, replicating the effects of old age at young NMJs. The current proposal will use a combination of highly-innovative methods to explore mechanistically the role of disrupted trophic factor signaling at the NMJ in old age. Our working hypothesis is that susceptibility to age-related neuromuscular dysfunction depends on motoneuron-muscle fiber trophic influences exerted by BDNF/TrkB signaling at the NMJ (aim 1) and motoneuron (aim 2). Furthermore, trophic factors can determine susceptibility to neuromuscular damage resulting from autophagy imbalance causing accumulation of protein aggregates and degeneration in old age. Two specific aims are proposed: Specific Aim 1) To determine the cellular (trophic factor dependent) mechanisms underlying susceptibility to neuromuscular dysfunction in old age. Specific Aim 2) To determine the molecular (trophic factor dependent) mechanisms underlying age-related effects at motoneurons. These results constitute the necessary foundation for the development of targeted therapies to mitigate aging effects on neuromuscular performance and increase the health span in the aging population with therapies initiated later in life to combat frailty and disability.

ABSTRACT The proposed studies exploit exciting new developments in neuroplasticity to enhance recovery of ventilatory- related diaphragm muscle (DIAm) activity following cervical spinal cord injury. There are nearly 17,000 new cases of spinal cord injury in the United States each year, with around 282,000 people affected. The majority of these injuries involve the cervical spinal cord and result in significant impairment of ventilatory-related DIAm activity and an inability to maintain adequate ventilation. Long-term dependence on mechanical ventilation is associated with significant morbidity and mortality. Thus, enhancing recovery of ventilatory-related DIAm activity following cervical spinal cord injury is highly significant. Upper-cervical (C1-C3) spinal cord injury disrupts direct excitatory descending bulbospinal glutamatergic (Glu) input to phrenic motor neurons (PhMNs). Importantly, most spinal cord injuries are incomplete, thus spared descending pathways to PhMNs are an extant substrate for neuroplasticity to restore DIAm activity, either by increasing excitatory (Glu) nerve terminal density and/or by altering postsynaptic Glu receptor (NMDA NR1) expression. In the proposed studies, we will employ a well-established C2 spinal hemisection (C2SH) model of incomplete spinal cord injury in rats, in which spontaneous recovery of ventilatory-related DIAm activity occurs in a BDNF/TrkB signaling-dependent fashion. Previously, we found that C2SH impairs ventilatory-related DIAm behaviors, which require recruitment of smaller (more excitable) PhMNs comprising fatigue resistant motor units. These ventilatory-related behaviors only partially recover over time, whereas, surprisingly, there is near full recovery of higher force airway clearance behaviors, which require recruitment of larger (less excitable) PhMNs comprising more fatigable motor units. The overall hypothesis of the proposed research is that the mechanisms underlying neuroplasticity and recovery of ventilatory-related DIAm activity after C2SH depend on PhMN size (more pronounced in smaller PhMNs), are mediated by NMDA Glu neurotransmission, and are promoted by BDNF/TrkB signaling. Three specific aims are proposed: 1) To determine the effect of BDNF/TrkB signaling on Glu presynaptic terminal density at PhMNs of differing size after C2SH; 2) To determine the effect of BDNF/TrkB signaling on NMDAR expression at PhMNs of differing size after C2SH; and 3) To determine whether NMDARs underlie the effects of BDNF/TrkB signaling on recovery of ventilatory-related DIAm activity after C2SH. The results of the proposed studies will guide development of effective therapeutic approaches to enhance recovery of respiratory function in patients with incomplete spinal cord injury.

The impact of acute airway inflammation is mediated by pro-inflammatory cytokines (e.g., TNF?), and underlies a number of respiratory diseases. A fundamental question is why are some individuals more susceptible than others to the negative impact of airway inflammation. We will explore a novel homeostatic mechanism, which protects airway smooth muscle (hASM) cells from the negative impact of inflammation-induced reactive oxygen species (ROS) formation and protein unfolding (endoplasmic reticulum (ER) stress). We believe that a failure in this homeostatic mechanism leads to increased ROS formation thereby exacerbating oxidative and ER stress. Overall Hypothesis: TNF?-induced ROS formation and protein unfolding activates the pIRE1?/XBP1s ER stress pathway in hASM, which initiates a homeostatic response directed towards increasing mitochondrial biogenesis and mitochondrial volume density to reduce O2 consumption and ROS formation by individual mitochondrion, while still meeting the increase in ATP demand ? sharing the energetic load across mitochondria. Furthermore, reduced Mfn2 disrupts mitochondrial tethering to the ER, thereby decreasing mitochondrial Ca2+ influx and maximum respiratory capacity of mitochondria. Aim 1: TNF?-induced activation of pIRE1?/XBP1s ER stress pathway increases mitochondrial volume density and reduces O2 consumption and ROS formation per mitochondrion. In hASM cells, the downstream impact of TNF?-induced activation of the pIRE1?/XBP1s ER stress pathway will be explored using transfection of a non-phosphorylatable IRE1? mutant plasmid (DP-IRE1?) or an unspliceable XBP1 (uXBP1) mRNA. In addition, we will examine the effects of siRNA knockdown of PGC1? and Mfn2 overexpression on TNF?-induced changes in mitochondrial biogenesis, mitochondrial volume density, O2 consumption and ROS formation. Aim 2: TNF?-induced reduction in Mfn2 disrupts mitochondrial tethering to ER, decreases mitochondrial Ca2+ influx and reduces maximum respiratory capacity of mitochondria. In hASM cells, we will examine the impact of DP-IRE1? or uXBP1 mRNA transfection and siRNA Mfn2 knockdown on TNF?-induced disruption of mitochondrial/ ER tethering, decreased mitochondrial Ca2+ influx and reduced maximum respiratory capacity of mitochondria. Aim 3: The impact of TNF? on activation of the pIRE1?/XBP1s ER stress pathway and downstream effects are mitigated by ROS scavenging and chemical chaperone treatment. In hASM cells, the mitigating effects of ROS scavenging and chemical chaperone treatment on TNF?-induced activation of the pIRE1?/XBP1s ER stress pathway will be examined.