Sieck, Gary C.

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

There is an age‐associated loss of phrenic motor neurons (PhMNs) and withdrawal of pre‐synaptic terminals at neuromuscular junctions (NMJs) in 24‐month old Fischer 344 (F344) rats. Previously, we showed that age‐related PhMN loss is concomitant with neuromuscular transmission failure and diaphragm muscle (DIAm) sarcopenia (an age‐related atrophy of muscle fibers and a decrease in specific force). It is unknown if pre‐synaptic terminal withdrawal precedes PhMN loss, which would implicate a reduction in a trophic influence emanating from muscle fibers (myogenic). We hypothesized that age‐related PhMN loss results from pre‐synaptic terminal withdrawal and reduced myogenic influence, which occurs predominantly at type IIx and/or IIb DIAm fibers (innervated by larger PhMNs). F344 rats were obtained from the National Institutes of Aging. Experimental ages were chosen to represent 100% and 50% survival at 6 months and 24 months‐old, respectively. PhMNs were first retrogradely labeled by intrapleural injection of cholera toxin‐subunit B (CTB), which is dependent on binding to pre‐synaptic terminal gangliosides. Thus, with pre‐synaptic terminal withdrawal, PhMNs would not be labeled. Subsequently (3 days after CTB), PhMNs were retrogradely labeled by sectioning the phrenic nerve and dipping the proximal end into a Rhodamine dextran (Rho) solution – this labeling is not dependent on pre‐synaptic terminals. CTB and Rho labeled PhMNs were visualized by confocal microscopy. Only optical slices containing the nucleus of Rho‐ and/or CTB‐labeled PhMNs were counted. We analyzed the differences in labeling of PhMNs by CTB and Rho in both age groups. In labeled PhMNs, short and long axis measurements were used to determine somal surface areas by assuming a prolate spheroid shape. In 6‐month old rats, there was no significant difference in the number of PhMNs labeled by CTB or Rho. In 24‐month old rats, the total number of labeled PhMNs was reduced compared to the 6‐month old rats, which primarily affected larger PhMNs. Furthermore, at 24‐months, fewer PhMNs were labeled with CTB compared to Rho and those labeled with Rho had larger somal surface areas. These results support our hypothesis that the age‐related loss of larger PhMNs results from pre‐synaptic terminal withdrawal and reduced myogenic influence. Support or Funding Information Support: NIH R01‐AG044615 American Physiological Society

Cardiac troponin I (cTnI) is a key regulator of cardiomyocyte contraction. Phosphorylation of cTnI at Ser22/23 is mediated by protein kinase A, C, and G, and is associated with reduced Ca 2+ sensitivity of myocardial contraction. In previous in vivo and in vitro studies, we showed that hypothermia/rewarming (rewarming shock) reduces the Ca 2+ sensitivity of myocardial contraction due to cTnI Ser22/23 phosphorylation. Rewarming shock was also found in isolated cardiomyocytes; however, demonstrating a relationship with cTnI phosphorylation is complicated by varying cardiomyocyte viability after isolation. Using a modified Langendorff technique, the cardiomyocyte viability gradually declines over a 4‐h period from ~80% to ~40%. This decline in cell viability is not accounted for in typical Western blot measurements and, therefore, assessments of cTnI phosphorylation are highly subject to misrepresenting actual physiological changes in cTnI phosphorylation. To address this issue, we developed a novel quantitative technique to measure cTnI phosphorylation at Ser22/23 in viable cardiomyocytes using confocal microscopy. We demonstrated that cardiomyocytes with a rod‐shaped morphology and uniform striation patterns display robust transient contractile and cytosolic Ca 2+ responses to electrical stimulation. All of these rod‐shaped cardiomyocytes exclude trypan blue, while a disproportionate fraction of non‐rod‐shaped cardiomyocytes are non‐viable. Separately, using 2D gel electrophoresis, we validated the specificity of an antibody that targets phosphorylated cTnI. Using this antibody, we verified that the extent of cTnI phosphorylation in rod‐shaped cardiomyocytes increased with isoproterenol in a dose dependent fashion. This novel methodology allows a more accurate measurement of cTnI phosphorylation in isolated primary cardiomyocytes that is not affected as cell viability declines over time. Support or Funding Information This publication was made possible by Grant Number T32 HL 105355 from the National Heart, Lung and Blood Institute (NHLBI), a component of the National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official view of NIH.

Inflammation and pro‐inflammatory cytokines (e.g., TNFα, IL‐13) induce airway smooth muscle (ASM) hyper‐reactivity and remodeling characteristic of asthma. We hypothesized that exposure to TNFα (and IL‐13) increases isometric force generation in porcine ASM via several underlying mechanisms: 1) increasing sensitivity to ACh stimulation; 2) enhancing the cytosolic Ca 2+ ([Ca 2+ ] cyt ) response to ACh; 3) increasing the Ca 2+ sensitivity of force generation; 4) enhancing the Ca 2+ ‐dependent phosphorylation of myosin regulator light chain (rMLC); 5) promoting expression of non‐muscle rMLC; 6) increasing actin and myosin heavy chain (MyHC) content (i.e., the number of contractile units); and/or 7) promoting cytoskeletal remodeling (actin polymerization). Studies were performed on intact ASM strips (~1 mm wide × ~5 mm long) dissected from porcine tracheas obtained from a local abattoir. The ASM strips were placed in PSS with and without TNFα (100 ng/ml) for 24 h. The ACh concentration dependence of force generation ASM was determined (ACh range from 10 nM to 1mM), and the EC50 (i.e., the ACh concentration that produces 50% of maximal ASM force) was determined. In control, untreated ASM, the EC50 for ACh was 2.6 μM compared to 1.3 μM following TNFα treatment. We found that across all ACh concentrations, ASM force increased after TNFα treatment with an ~50% increase in maximum ASM force. The [Ca 2+ ] cyt response induced by the EC50 [ACh] was similar in control and TNFα treated ASM. Thus, with an increase in force and no effect on [Ca 2+ ] cyt , there was an apparent increase in Ca 2+ sensitivity of force after TNFα treatment. However, in both control and TNFα treated ASM, the extent of rMLC phosphorylation induced by ACh stimulation was not affected by TNFα treatment. Importantly, the extent of rMLC phosphorylation at the ACh EC50 was comparable between control and TNFα treatment indicating no change in Ca 2+ sensitivity. The expression of non‐muscle rMLC (determined by 2D gel electrophoresis) was also unaffected by TNFα treatment. Total actin and MyHC concentrations increased by ~45% following TNFα treatment; thus, the number of contractile units contributing to force increased. This was reflected by a marked increase in the Ca 2+ activation of ATP consumption (cross‐bridge cycling) of ASM following TNFα treatment. Cytoskeletal remodeling was evaluated by ACh‐induced actin polymerization (F‐/G‐actin ratio) but was found to be unaffected by TNFa treatment. Together, our results show that two primary mechanisms underlie the marked increase in ASM force generation induced by 24‐h TNFα exposure: 1) an increase in ACh sensitivity, and 2) an increase in actin and MyHC concentrations (number of contractile units). Support or Funding Information Supported by NIH grant HL126451 (GCS) and HL150890 (GCS)

Cytoskeletal remodeling in smooth muscle occurs with activation and involves actin polymerization. Remodeling of the cortical cytoskeleton allows tethering of contractile proteins and force transmission to the extracellular matrix. Smooth muscle contraction involves cross‐bridge cycling and ATP hydrolysis, which are both sensitive to loading. During isometric activation, the rate of ATP hydrolysis is initially high but declines with time reaching a steady‐state, while peak force is maintained. Thus, tension cost, i.e., the rate of ATP hydrolysis per unit of force, decreases over time, reflecting increased efficiency of smooth muscle activation. We hypothesize that the time‐dependent decrease in ATP hydrolysis rate and tension cost is due to cytoskeletal remodeling and an increase in internal loading on cross‐bridges. In permeabilized porcine airway smooth muscle (ASM) strips, isometric force and ATP hydrolysis rate were simultaneously measured after maximum Ca 2+ activation (pCa 4.0). From each animal, two ASM strips were tested: 1) Inhibition of actin polymerization and cytoskeletal remodeling using Cytochalasin‐D (Cyto‐D; treated (1 μM for 30 min); and 2) Untreated controls. Compared to untreated controls, Cyto‐D‐induced inhibition of actin polymerization reduced both peak and steady‐state isometric force by ~40%, while peak and steady‐state ATP hydrolysis rate increased by ~30% and ~20%, respectively. As a result, at peak and steady‐state, tension cost increased by ~100% and ~60%, respectively, following Cyto‐D treatment. These results support our hypothesis that the time‐dependent decrease in ATP hydrolysis rate reflects an increase in internal load on cross‐bridges via cytoskeletal remodeling. Cyto‐D inhibition of cytoskeletal remodeling impairs contractile protein tethering and thus reduces internal loading, resulting in faster cross‐bridge cycling and increased ATP hydrolysis rate. Support or Funding Information Supported by NIH grant HL126451(GCS), HL150890 (GCS) and the Mayo Clinic

Inflammation triggers a homeostatic response that is mediated by pro‐inflammatory cytokines such as TNFα. We hypothesize that in a motor neuron‐like cell line, NSC‐34, TNFα decreases mitochondrial function, specifically succinate dehydrogenase (SDH) activity, which is a key enzyme in the tricarboxylic acid (TCA) cycle as well as complex II in the electron transport chain (ETC). Experiments were performed on cultured NSC‐34 cells that were differentiated by serum depletion until neurite structures (>50 μm) were observed. The differentiated cells were treated with TNFα (20 ng/ml) for 24 h. To measure the maximum velocity of the SDH reaction (SDH max ), cells were incubated in a solution containing 80 mM succinate and 1.5 mM nitro blue tetrazolium (NBT), the reaction indicator. As the SDH reaction proceeded, images were acquired every 15 s using a 40× objective on an Olympus IX71 inverted microscope. Linearity of the SDH reaction was confirmed across the 8‐min period, and SDH max was determined and expressed as mM fumarate/L tissue/min. To determine mitochondrial volume density, the cells were loaded with MitoTracker Green and imaged in 3D using a confocal microscope. The cell volume occupied by labeled mitochondria was determined from 3D reconstructions of 1 mm optical slices. In NSC‐34 cells exposed to TNFα, we found that mitochondrial volume density increased, and mitochondria were fragmented as compared to untreated controls. We also found that TNFα exposure decreased SDH max when normalized for mitochondrial volume density. In other cell types, we found that TNFα‐induces an increase in PGC1a and Drp1 expression and a decrease in Mfn2 expression, all of which affect mitochondrial structure. In related studies, we also found that TNFα exposure decreases O 2 consumption per mitochondrion in NSC‐34 cells. We conclude that by reducing the redox potential (SDH max ) of mitochondria, O 2 consumption per mitochondrion is reduced, thereby damping ROS formation. The impact of this homeostatic response is offset by mitochondrial biogenesis and an increase in mitochondrial volume density, thereby maintaining overall cellular O 2 consumption and ATP production but at reduced cost of ROS formation. Support or Funding Information Supported by NIH grants AG44615 and HL105355.

Neuroinflammation triggers a homeostatic response that is mediated by pro‐inflammatory cytokines such as TNFα. We hypothesize that in a motor neuron‐like cell line, NSC‐34, TNFα decreases mitochondrial O 2 consumption. NSC‐34 cells were differentiated by serum depletion (~24 h) until neurite structures (>50 μm) were observed. The cells were then exposed to TNFα (20 ng/ml) for 24 h. To determine mitochondrial volume density, the cells were loaded with MitoTracker Green and imaged in 3D using a Nikon A1R confocal microscope (60x oil immersion 1.4 NA). After treatment with TNFα, mitochondria appeared fragmented but mitochondrial volume density increased by ~50%, as compared to control untreated cells. A Agilent Seahorse XF Analyzer was used to determine the O 2 consumption of treated and untreated cells during a stress test that determines basal respiration, ATP production and maximum respiration. O 2 consumption was normalized to mitochondrial volume density in both treated and untreated groups. After TNFα exposure, basal respiration, ATP production and maximum respiration decreased per mitochondrion. However, overall O 2 consumption of the cells was unchanged after TNFα exposure due to an offsetting increase in mitochondrial volume density. Using MitoSox, we also measured reactive oxygen species (ROS) formation in treated and untreated NSC‐34 cells. ROS is a byproduct of mitochondrial O 2 consumption, and can cause cellular damage. After TNFα exposure, ROS formation increased compared to untreated cells. We conclude that mitochondrial biogenesis and an increase in mitochondrial volume density represent a homeostatic mechanism to reduce O 2 consumption the per mitochondrion, thereby damping ROS formation while maintaining overall cellular O 2 consumption and ATP production. Support or Funding Information Supported by NIH grant AG44615 and HL105355.

Nerve-muscle interaction is poorly understood in the context of early-onset spasticity and hypertonia. In an animal model of early-onset spasticity, spa mice, we found a marked impairment of tibialis anterior neuromuscular transmission. This impairment is associated with an increased innervation ratio (mean number of muscle fibers innervated by a single motor neuron). These disturbances may underlie weakness and gait disturbances observed in individual with developmental hypertonia and spasticity.

In previous studies, we found that after cervical spinal cord hemisection at C2 (SH), neuromuscular transmission improved in the rat diaphragm muscle (DIAm). We also found that exogenous brain‐derived neurotrophic factor (BDNF) treatment enhanced neuromuscular transmission, whereas inhibiting activation of its receptor (TrkB) worsened neuromuscular transmission. We hypothesized that expression of BDNF and/or its receptor TrkB at phrenic motoneurons would increase following SH. In the present study, adult male rats underwent SH or sham surgery. Phrenic motoneurons were labeled by injection of cholera toxin B into the DIAm and retrogradely‐labeled phrenic motoneurons were sampled by laser capture microdissection (LCM). BDNF and TrkB receptor (truncated – T1 and full length – FL) mRNA were measured in LCM‐microdissected motoneurons using quantitative real‐time RT‐PCR and expressed as a ratio to ribosomal S16 RNA. We found that BDNF mRNA expression in phrenic motoneurons increased by 3 days after SH (~2‐fold). Similarly, both TrkB.T1 and TrkB.FL mRNA increased by 3 days after SH (~17‐ and ~11‐fold, respectively). It is possible that the relative expression of neurotrophins and their receptors (truncated or full‐length) may determine the time‐course of restoration of phrenic activity following SH. Supported by NIH grants AR51173 and HL37680 and the Mayo Foundation.