Sieck, Gary C.

Muscle protein content reflects a balance between protein synthesis and degradation, which are likely regulated by neuromuscular activity and/or neurotrophin influence. In previous studies, we found that unilateral denervation (DNV) of the rat diaphragm muscle (DIAm) decreases myosin heavy chain (MHC) protein content after approximately 7 days. However, we did not determine whether the decrease in DIAm MHC protein content was the result of a decrease in protein synthesis, an increase in protein degradation, or both. We hypothesize that protein degradation plays a significant role in the decrease in DIAm protein content following DNV. In this study, we analyzed the ubiquitination state of cellular proteins as a marker of protein degradation. Conjugation of ubiquitin to cellular proteins targets proteins for degradation by proteosomes. We analyzed DIAm from adult male Sprague‐Dawley rats 3, 7 and 14 days after DNV. The DIAm was homogenized and protein ubiquitination was analyzed by Western analysis. No difference in ubiquitination was detected between control (CTL), sham surgery‐treated (sham), and 3‐day DNV animals. However, following 7 and 14 days of DNV, there was an approximate five‐fold increase in protein ubiquitination in DIAm compared to CTL and sham groups. These results demonstrate that DIAm protein ubiquitination increases between 3 and 7 days following DNV, and support the hypothesis that protein degradation contributes to the DNV‐induced decrease in DIAm MHC protein content. Supported by NIH grants AR51173 and HL37680.

Diseases of the central nervous system commonly affect motoneurons. In particular, spinal cord injury, aging and neurological disorders can cause motoneuron dysfunction or loss. There are no effective diagnostic tools or treatments that specifically target motoneurons. We hypothesized that nanoparticles can be used to target drug delivery to motoneurons. Specifically, mesoporous silica nanoparticles (MSNPs) may be ideal since they can be independently modified allowing loading of various cargos and surface‐engineered for bio‐functionality. The aim of this study was to characterize the role of nanoparticle size, concentration, time of incubation and surface coating on uptake by cultured motoneurons. Four different fluorescently‐labeled MSNPs were evaluated: PEG‐PEI‐Ace MSNPs of 150 nm and 50 nm, lipid coated MSNPs (protocell ‐ 140 nm) and PEG‐PEI MSNPs (50nm). PEG‐PEI‐Ace showed significant greater motoneuron uptake (p<0.05) after longer periods of incubation (12h for 50 nm & 24h for 150 nm). Protocells and PEG‐PEI achieved maximal uptake by 3h. Differentiated motoneurons displayed significant uptake at cell soma and dendrites. Regardless of cell density and duration of treatment, there was minimal evidence of cytotoxicity by any MSNP. In conclusion, MSNPs can be used to target delivery to motoneurons. Specifically, coating, size and concentration affect motoneuron uptake. Smaller and coated PEG‐PEI‐Ace MSNPs showed substantial uptake by motoneurons. These results may help inform the appropriate characteristics of MSNPs for specific in vivo application.

The force within a muscle may be assessed using intramuscular pressure measurements (IMP). However, a limiting factor in implementing this technology in the clinic is the observed variability of IMP measurements. The variability may be due in part to the flow of interstitial fluid. The objective of this study was to develop a technique for tracking interstitial fluid flow during muscle lengthening using fluorescent microspheres. Additionally, the effect of microsphere diameter on dispersion was evaluated. Fluorescent microspheres were injected into the rat tibialis anterior (TA) either before (right limb) or after (left limb) lengthening to 10% strain (n = 5). To help discriminate the effects of microsphere size, 10 µl of microspheres (0.25% by volume) were injected simultaneously: 0.1 µm in sky blue and 0.2 µm in yellow green. Once frozen, 20 µm muscle cross‐sections were stained and imaged using confocal microscopy to evaluate the microsphere's axial dispersion as a percent of muscle length. The microsphere dispersion was normalized to the muscle length. The fluorescent microspheres flowed with the interstitial fluid and did not penetrate the muscle fiber. There was significantly greater microsphere dispersion with passive strain (p‐value > 0.01) for lengthened samples (31±7%) compared to static samples (17±9%). Dispersion did not differ for the two bead sizes. In conclusion, this illustrates the microspheres track the interstitial fluid and passive lengthening results in additional dispersion. Supported by NIH under award number R01HD31476 and T32HL105355.

Sepsis is associated with microvascular hyporeactivity to agonists and may be reflective of reduced Ca 2+ ‐sensitivity within smooth muscle cells. We hypothesize that impaired vasoreactivity during LPS‐mediated inflammation is associated with decreased Ca 2+ sensitivity in small mesenteric resistance arteries (SMRA). SMRAs (190–220 μm) isolated from LPS‐treated (15 mg/kg ip, 18 hours) and saline‐treated control mice were mounted on a pressure myograph, superfused, and loaded with fura‐2. Arteriolar diameter and global intracellular Ca 2+ were simultaneously measured. Smooth muscle Ca 2+ ‐sensitivity of SMRAs was assessed by stepwise increases in extracellular Ca 2+ (0 to 2 mM) under depolarizing conditions (120 mM K + ). SMRA isolated from mice treated with LPS demonstrated hyporesponsiveness to Ca 2+ . Extracellular mediated Ca 2+ vasoconstriction in intact vessels resulted in an increase in EC 50 (0.23 ± 0.01 mM vs 0.39 ± 0.03 mM∗) and a reduction in E max (42.8± 1.1% vs 26.6 ± 3.4%∗). Removal of the endothelium resulted in near normal response to Ca 2+ . In intact vessels, LPS treatment decreases vascular smooth muscle Ca 2+ sensitivity as compared to controls. NOS inhibition (L‐NNA, 1 μM) restores responsiveness to Ca 2+ without restoring Ca 2+ sensitivity. LPS‐induced inflammation results in NO‐independent decrease in Ca 2+ ‐sensitivity within vascular smooth muscle cells.

By binding to a nebulin site on actin in skeletal myofibrils phalloidin (PH) causes a force decrease (phase 1) followed by an increase in force (phase 2). The initial force decrease likely reflects a disturbance in actin‐nebulin interaction. The aim of this work was to determine the role of myosin cross‐bridges (XBs) and Ca 2+ in PH‐induced force decrease in skinned rabbit psoas fibers. Force response to PH was studied in the presence of 30 mM 2,3‐Butanedione monoxime (an agent reducing a fraction of strongly bound XBs), where we found that the phase 2 response was suppressed and PH induced 60–70% force decrease. Pretreatment with PH of relaxed (pCa 9, strongly bound XBs are absent) or rigor (in the absence of ATP, all XBs are strongly bound to actin) muscles also resulted in force loss. At the same duration of pretreatment, the remaining force was higher and more sensitive to PH in the latter case. The same properties were found for dethiophalloidin (DTPH), where it was also shown that the force loss was essentially the same at pCa 4 and pCa 9. We concluded that rigor XBs, but not Ca 2+ , affect the rate of PH binding to actin in myofibrils. The XBs mediated changes in nebulin site on actin are supportive for suggested regulatory nebulin role. Supported by NIH grants HL68555 and HL37680.

Rewarming shock following accidental hypothermia is a potentially fatal scenario involving cardiac dysfunction. Previously our lab demonstrated that rewarming shock results in a decrease in Ca 2+ sensitivity of contraction in cardiac muscle due to an increase in cTnI phosphorylation. In this study, we hypothesized that rewarming shock initiates a cascade of intracellular events including reactive oxygen species (ROS) formation, endoplasmic reticulum (ER) stress, and mitochondrial disruption. Isolated cardiomyocytes from Sprague‐Dawley rats were exposed to a hypothermia/rewarming protocol (cooled for 1 h, held at 17 o C for 30 min, rewarmed for 1 h) that results in contractile dysfunction indicated by an increase in cytosolic Ca 2+ and reduced cell shortening measured using an IonOptix system. Rewarming shock increased ROS levels measured by an HPLC‐based dihydroethidium assay and by MitoSox fluorescence. Hypothermia/rewarming was also associated with ER stress indicated by increased expression of ER stress markers (Grp78, PERK, and CHOP) measured by Western blot. Finally, rewarming shock was associated with mitochondrial disruption evidenced by mitochondrial membrane depolarization (decreased in TMRM fluorescence) and by an increase in cytosolic cytochrome c (measured by Western blot). Our results support our overall hypothesis and suggest that these ER and mitochondrial intracellular events may underlie myocardial contractile dysfunction following hypothermia/rewarming. Supported by NIH Grant 5T32HL105355‐02 (GCS)

Sarcopenia is the age‐related decline of skeletal muscle function. Sarcopenia involves loss of motor neurons and the subsequent denervation of muscle fibers. Denervated muscle fibers can be re‐innervated through terminal axonal sprouting of neighboring axons. Given the predominant role of motor neurons in establishing muscle fiber type, we hypothesized that sarcopenia (and the associated partial denervatin effects) result in clustering of muscle fibers of the same type (classified by myosin heavy chain isoform composition). Fiber type specific immunofluorescence histology was used to quantify fiber clustering in murine diaphragm muscle (n=15) at 6 and 24 months of age. Clustering was analyzed using the distance between fibers of the same fiber type as well as quantification of the number of type‐identified fibers in a cluster. There was a 14% decrease to the closest fiber for type I and a 24% increase for type IIx and/or IIb fibers (P<0.001) in old vs. young mice. Additionally, a 34% increase to the 3 closest type IIx and/or IIb fibers was found (P<0.001). Changes in the interfiber distances for type‐identified fibers reveals important effects that consider changes in fiber size and proportion across disease conditions. This novel method of analyzing fiber type clustering may be useful in examining pathophysiological conditions of motor unit loss in neuromuscular disorders, myopathies, dystrophies, injuries, or amyotrophic lateral sclerosis. Supported by R01‐AG044615, T32‐HL105355, and the Mayo Clinic.

The diaphragm muscle (DIAm) must be able to generate sufficient forces to sustain ventilatory and non‐ventilatory behaviors throughout the lifespan. Age‐related decreases in DIAm fiber cross‐sectional area and specific force, i.e., sarcopenia, may impair the ability to generate transdiaphragmatic pressures (Pdi) necessary to accomplish expulsive, non‐ventilatory behaviors essential for airway clearance. We examined the effects of aging on DIAm function in both male and female mice at 6 and 24 months of age (n=79). Maximum DIAm specific force was 30% lower in old mice (P蠄0.001) and Pdi generated during non‐ventilatory behaviors was ~40% less in old mice (P蠄0.001). No differences in either DIAm force or Pdi were found between males and females. Collectively, these results indicate that DIAm sarcopenia is present in both sexes and significantly affects the ability to generate Pdi needed for airway clearance in old age. Supported by R01‐AG044615, T32‐HL105355, and the Mayo Clinic.

Motor units are normally recruited in an orderly manner in order to accomplish ventilatory behaviors. The duration of non‐stationarity in electromyographic (EMG) bursts provides an estimate of the period of motor unit recruitment. Motor unit recruitment also reflects central drive, which can be estimated by the root‐mean‐squared (RMS) EMG value 75 ms after the EMG burst onset (RMS 75 ). Following unilateral diaphragm muscle (DIAm) paralysis induced by upper cervical spinal cord hemisection at C 2 (SH), there is gradual spontaneous recovery of ipsilateral EMG activity. Increased activity of the contralateral (uninjured) DIAm is sufficient to maintain ventilation following SH. We hypothesized that changes in central drive can be estimated by the period of non‐stationarity and RMS 75 from the contralateral DIAm EMG following ipsilateral paralysis and spontaneous recovery of DIAm EMG activity. We found that the duration of non‐stationarity increased to a greater extent in rats that did not recover ipsilateral EMG activity by 14 days post‐SH (D14) compared to those that did. In addition, we found that RMS 75 values following SH were reduced in rats displaying recovery of ipsilateral DIAm EMG activity compared to rats that did not. Collectively, these results suggest that the duration of non‐stationarity and RMS 75 can be used to evaluate recovery of ipsilateral DIAm function following spinal cord injury.