Sieck, Gary C.

The diaphragm muscle (DIAm) comprises a mixed population of motor units, with FInt and FF units (type IIx and/or IIb fibers) vulnerable to sarcopenia ‐ age‐associated reductions of specific force and cross‐sectional area (CSA). Fatigue‐resistant S and FR units (type I and IIa fibers) are relatively spared. There is controversy as to whether aging confers an enhanced resistance to DIAm fatigue, with interpretations relying on relative measurements of force. We assessed the loss of DIAm force in 6‐ and 24‐month old Fischer 344 rats at 10, 40 and 75 Hz stimulation in 330 ms duration trains repeated each s (33% duty cycle) across a 2 min period. Although the initial specific force of the DIAm was lower in older rats across all stimulation frequencies, the final residual DIAm specific force after 2 min of stimulation was the same (~5 N/cm 2 ) at all ages. This gave the misconception of an improved fatigue resistance, especially at 40 and 75 Hz. We also found that aging is associated with an increase in the relative contributions of type I and IIa fiber CSA to DIAm mass with decreased contributions of type IIx and/or IIb fibers. When these changing contributions are factored into a fiber type‐specific model of DIAm force generation, we found that ventilatory behaviors require activation of only type I and IIa fibers, regardless of age or fatigue. By contrast, the model predicts that aging impairs the ability of the DIAm to effectively perform expulsive manoeuvres, even in the non‐fatigued condition. In summary, relative fatigue measurements are not indicative of improved fatigue resistance in aging and are an artifact of reduced initial specific force contributions from FInt and FF units. These results are consistent with previous studies showing the relative vulnerability of FInt and FF units and conserved ventilatory behaviors in aging DIAm. Support or Funding Information R01‐AG044615 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

In cardiomyocytes, the endoplasmic reticulum (ER) plays a dynamic role in excitation‐contraction coupling by regulating cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ). Under pathological conditions the ER accumulates unfolded proteins triggering a homeostatic ER stress response to minimize and limit protein unfolding (via chaperone proteins) and accumulation (by decreasing synthesis and increasing degradation). The interactions between ER stress, [Ca 2+ ] cyt regulation and downstream contractile responses remain unknown, although such interactions may underlie the etiology of various cardiovascular diseases. We hypothesized that in cardiomyocytes ER stress alters the regulation of [Ca 2+ ] cyt and reduces cardiac contractility by decreasing myofilament Ca 2+ sensitivity. In isolated rat cardiomyocytes, ER stress was induced by tunicamycin, a naturally occurring antibiotic that induces ER stress by inhibiting N‐linked glycosylation. In one set of experiments, we used Western blotting techniques to evaluate the effects of ER stress on phosphorylation of cTnI at Ser23/24. In a second set of experiments, we evaluated the impact of tunicamycin on excitation‐contraction coupling by simultaneously measuring [Ca 2+ ] cyt (using Fura‐2 AM ratiometric imaging) and contractile (sarcomere length (SL) shortening) responses to electrical field stimulation using an IonOptix system. Tunicamycin induced an increase in phosphorylation of cTnI by 3–6 h. The evoked [Ca 2+ ] cyt transient was relatively unaffected by tunicamycin while the contractile response was markedly reduced. This resulted in a rightward shift in the phase loop relationship between [Ca 2+ ] cyt and SL shortening indicating reduced myofilament Ca 2+ sensitivity. We conclude that ER stress may underlie contractile dysfunction in a variety of cardiac diseases. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Excitatory descending glutamatergic (Glu) synaptic input to phrenic motor neurons (PhMNs) emanates from the ventrolateral medulla and is responsible for inspiratory drive and a range of higher force behaviors. Disruption of this excitatory input following unilateral C2 hemisection (C2SH) results in cessation of inspiratory‐related diaphragm muscle (DIAm) activity ipsilateral to injury, but DIAm activity during higher force behaviors persists, reflecting the contribution of spared contralateral inputs. In a model of PhMN (DIAm motor unit) recruitment, we proposed that smaller PhMNs innervating more fatigue resistant DIAm motor units are recruited to accomplish ventilatory behaviors, whereas larger PhMNs innervating more fatigable motor units are recruited to accomplish more forceful expulsive and straining behaviors. We hypothesized that differences in the distribution of ipsilateral and contralateral Glu synaptic inputs to PhMNs contribute to the effects of C2SH on the range of motor behaviors accomplished by the DIAm. Accordingly, we hypothesized that C2SH primarily disrupts ipsilateral Glu synaptic inputs to smaller PhMNs, whereas Glu synaptic inputs to larger PhMNs are minimally impacted after C2SH. To address this hypothesis, we examined changes in Glu presynaptic input onto retrogradely labeled PhMNs using immunohistochemistry for VGLUT1 and VGLUT2. We found that following C2SH (by 7 days), there was a ~45% reduction in ipsilateral Glu synaptic input to PhMNs compared to a ~20% reduction contralaterally. In addition, ipsilateral to C2SH, Glu synaptic input to smaller PhMNs was reduced to a greater extent compared to that to larger PhMNs (55 vs. 35% reduction, respectively). Contralateral to C2SH, inputs to PhMNs were similarly reduced independent of motor neuron size. These results are consistent with more uniformly distributed contralateral inputs to all PhMNs and predominant input to smaller PhMNs ipsilaterally. These results also indicate that the source of Glu synaptic input to PhMNs varies depending on motor neuron size and likely reflects different motor control, including possibly distinct central pattern generator and premotor circuits. Support or Funding Information Grant Support: NIH Grant R01 HL96750 and Mayo Clinic Graduate School of Biomedical Sciences This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Brain‐derived neurotrophic factor (BDNF) binds with high affinity to tropomyosin‐related kinase receptor subtype B (TrkB), and plays an important role in neuromuscular junction (NMJ) function (regulating neuromuscular transmission and muscle innervation). We previously showed impaired neuromuscular transmission following acute inhibition of TrkB kinase activity in TrkB F616A mice harboring a point mutation that renders the kinase domain susceptible to inhibition by 1NMPP1 treatment. Neuromuscular transmission failure is expected to predominantly affect the ability of more fatigable motor units to generate force and thus primarily impair higher force motor behaviors. Accordingly, we hypothesized that acute administration of 1NMPP1 will impair the ability of the diaphragm muscle to accomplish higher force behaviors in adult, 6‐month old TrkB F616A mice. Transdiaphragmatic pressure (Pdi) measurements were conducted across a range of motor behaviors in lightly anesthetized mice during eupnea, exposure to hypoxia (10% O 2 )/hypercapnia (5% CO 2 ), tracheal occlusion, spontaneous deep breaths (“sighs”) and during maximal activation elicited by bilateral phrenic nerve stimulation. Adult male and female TrkB F616A mice at 6 months were injected with 1NMPP1 or vehicle (DMSO) one hour prior to Pdi measurements. The Pdi generated during eupnea and hypoxia/hypercapnia was ~8 cm H 2 0 compared to ~70 cm H 2 0 during maximal stimulation. There was a significant reduction in Pdi during maximal force with bilateral phrenic nerve stimulation in the 1NMPP1 group compared to the vehicle control (p=0.01). There was no effect of 1NMPP1 on Pdi during eupnea, hypoxia/hypercapnia, tracheal occlusion, or sigh behaviors. The reduction in Pdi is generally similar to the impairment in neuromuscular transmission consistent with a predominant effect on motor units innervating fast‐twitch fatigable muscle fibers in the diaphragm muscle. Thus, impaired trophic BDNF/TrkB signaling can be expected to limit the range of motor behaviors accomplished by the diaphragm muscle and may be an important contributor to neuromuscular dysfunction. Support or Funding Information This study was supported by NIH grant R01 AG057052. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Brain derived neurotrophic factor (BDNF) acting through the high affinity tropomyosin‐related kinase receptor B (TrkB) plays an important role in synaptic plasticity and function at the neuromuscular junction (NMJ) as well as maintenance of neuromuscular transmission. Our previous studies showed that susceptibility to age‐related changes in diaphragm muscle (DIAm) NMJ morphology and denervation depends on trophic influences exerted by BDNF/TrkB signaling. Indeed, TrkB kinase activity is necessary to stabilize DIAm NMJ pre‐synaptic terminals in 18 month old mice (representing early old age; ~90% survival). However, by 24 months of age (~75% survival), BDNF/TrkB signaling has no effect on DIAm neuromuscular transmission, suggesting reduced TrkB kinase activity in the older age groups. We hypothesized that the NMJs that are resilient to old age do not depend on TrkB kinase activity for stabilization of pre‐synaptic terminals. Male and female 24 month old TrkB F616A mice were used to examine the structural properties of NMJs across various muscles with varying susceptibility to age‐related effects (DIAm, EDLm, and SOLm). Confocal microscopy was used to compare pre‐ and post‐synaptic morphology and denervation following a 7 day treatment with the phosphoprotein phosphatase‐1 derivative 1NMPP1, which inhibits TrkB kinase activity in TrkB F616A mice vs. vehicle treatment. Inhibition of TrkB kinase activity had no effect on pre‐synaptic or post‐synaptic terminal volume in DIAm, EDLm, and SOLm. Similarly, the overlap of pre‐ and post‐synaptic structures and the proportion of denervated NMJs did not change with 1NMPP1 treatment in all muscle types. The average percent of denervated NMJs was 8, 22, and 7% in DIAm, EDLm, and SOLm, respectively. Collectively these results suggest that the requirement of TrkB kinase activity for stabilization of NMJs in DIAm, EDLm and SOLm wanes in old age. Support or Funding Information Supported by R01‐AG057052. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Airway inflammation leads to increased intracellular Ca 2+ ([Ca 2+ ] i ) in airway smooth muscle (ASM). There is now growing evidence that the bidrectional plasma membrane Na + /Ca 2+ exchange (NCX) may contribute to ASM [Ca 2+ ] i regulation. However, the roles of NCX‐mediated Ca 2+ influx vs. efflux, or the role of NCX in airway inflammation are largely unknown. In the present study, we examined NCX expression and function in human ASM exposed to the pro‐inflammatory cytokines TNFα and IL‐13. Western blot analysis of human ASM tissue showed significant expression of the NCX1 isoform, with substantial increase in expression following overnight cytokine exposure. In fura‐2 loaded human ASM cells, NCX‐mediated Ca 2+ influx as well as efflux (measured as slopes of changes in [Ca 2+ ] i ) was elicited using techniques previously described for cardiac muscle. Contribution of NCX was verified by measuring [Na + ] i using the fluorescent Na + indicator SBFI. NCX‐mediated influx was verified by demonstrating prevention of rising [Ca 2+ ] i or falling [Na + ] i in the presence of the NCX inhibitor KBR7943. In second set of studies, NCX siRNA transfection of human ASM substantially blunted efflux and influx mode of NCX (siRNA effect verified by Western blot). Exposure to TNF‐α as well as IL‐13 significantly enhanced influx mode of NCX. Although both cytokines also increased efflux mode of NCX, these effects were relatively smaller. These data suggest that NCX may be one mechanism contributing to inflammation‐induced enhancement of [Ca 2+ ] i. Supported by NIH grants HL74309, RR024150, and the Flight Attendants Medical Research Institute (FAMRI). Additional funding from the Department of Anesthesiology, Mayo Clinic.

In skeletal muscles, force gradation depends on the level of motor unit (MU) activation. MU recruitment generally follows a specific order, with MUs comprising type I or type IIa fibers more active than type IIx and/or IIb. Direct evidence of MU recruitment across motor behaviors is limited to samples of relatively few MUs within a pool. The purpose of this study was to determine the extent of diaphragm MU recruitment across different motor behaviors. We hypothesized that in vivo labeling of individual neuromuscular junctions (NMJs) by FM4‐64 reflects the activation of MUs . Adult rats were exposed to eupneic (E), hypoxic/hypercarbic (H) conditions or supramaximal phrenic nerve stimulation (S). Individual diaphragm NMJs were type‐identified by myosin heavy chain expression. Average FM4‐64 labeling for NMJs at type I or IIa fibers vs. type IIx and/or IIb fibers increased progressively from E to H to S. The proportion of labeled NMJs of each type also increased significantly. At type I or IIa fibers, 40%, 59% and 90% of NMJs were labeled during E, H and S, respectively. At type IIx and/or IIb fibers, 21%, 32% and 82% of NMJs were labeled during E, H and S, respectively. Thus, using an in vivo labeling method there is direct evidence of progressive MU recruitment across different ventilatory and non‐ventilatory behaviors in the rat diaphragm. Supported by NIH grants HL37680 and AR51173, and the Mayo Clinic.

Spinal cord hemisection at C2 (SH) disrupts descending drive to ipsilateral phrenic motoneurons (PMNs) causing unilateral diaphragm muscle (DIAm) paralysis. Previously, we found a decrease in motoneuron size at 2 weeks post‐SH. Recently, we found neuregulin (NRG) in synaptic boutons surrounding PMNs. NRG is a trophic factor known to regulate development of the neuromuscular junction, but the effect of NRG on motoneurons is unknown. We hypothesized that 1) NRG input to PMNs is supraspinal in origin, and 2) SH disrupts NRG input to PMNs. Adult male Sprague‐Dawley rats underwent SH or sham‐surgery. PMNs were retrogradely labeled by intramuscular injection of Alexa488‐conjugated cholera toxin B. Using laser capture microdissection, we found that PMNs express mRNA for the NRG receptors ErbB2 and ErbB3, providing a functional substrate for a NRG effect. Two weeks post‐SH, labeled PMNs and NRG boutons were imaged using confocal microscopy. PMN morphology and the number of NRG boutons in the vicinity of PMNs were determined using a computerized, semi‐automated reconstruction algorithm. NRG boutons were fairly homogeneously distributed on motoneurons independent of size. Following SH, few NRG boutons were observed. We conclude that 1) NRG input to PMNs derives from a supraspinal source; and 2) SH removes NRG input to PMNs. It is possible that NRG input exerts a trophic influence on PMNs. Supported by NIH grants HL37680, AR51173 and Mayo Clinic

In airway smooth muscle (ASM), the state of sarcoplasmic reticulum (SR) Ca 2+ stores is an important determinant of [Ca 2+ ] i response to agonist. We have previously shown that acetylcholine (ACh) induces sustained [Ca 2+ ] i oscillations representing repetitive SR Ca 2+ release and reuptake. Thus, SR refilling is a key component in refilling of SR stores. We hypothesized that, like cardiac and vascular smooth muscle, ASM SR refilling is regulated by calmodulin (CaM)‐CaM kinase (CaMKII) dependent phosphorylation of phospholamban (PLB). In ASM cells from fresh porcine trachea, effects of W7 (CaM antagonist) and KN‐93/KN‐62 (CaMKII antagonists) on [Ca 2+ ] i responses to ACh were measured using fluorescence microscopy. Separately, SR‐enriched membranes were isolated to monitor ATP‐energized Ca 2+ uptake and in vitro phosphorylation of PLB in the presence of CaMKII inhibitors. KN‐93 significantly slowed decay of [Ca 2+ ] i responses, and slowed or inhibited ongoing [Ca 2+ ] i oscillations, with visible slowing of fall‐times of individual oscillations. Pre‐exposure to W7 or KN‐62 did not prevent initiation of oscillations. In SR‐enriched membranes, addition of KN‐93/KN‐62 resulted in significantly slower Ca 2+ uptake, accompanied by a matching decrement in the in vitro phosphorylation of Thr 17 ‐PLB, indicating the influence of CaM‐CaMKII on this protein. These data suggest that agonist‐induced [Ca 2+ ] i regulation in ASM involves the CaM‐CaMKII pathway which indirectly influences Ca 2+ release and reuptake, likely through PLB. Supported by NIH grant HL74309 (GCS)