C. George Carlson

Chronic treatment with agents that stabilize cytosolic IκB-α enhances survival and improves resting membrane potential in MDX muscle fibers subjected to chronic passive stretch

The potential pathogenic role of increased NFkappaB signaling in passively stretched dystrophic skeletal muscle was examined by treating adult mdx mice with an agent that stabilized cytosolic IkappaB-alpha (pyrollidine dithiocarbamate, PDTC)and examining the effects of this treatment on the chronically stretched mdx triangularis sterni (TS) muscle. Daily PDTC treatment significantly increased the number of surviving striated TS fibers regardless of age. TS fibers from untreated mdx mice had significantly lower resting potentials (RPs) than nondystrophic mice. Treatment with GdCl3 to block resting Ca2+ influx had no effect on RP in either nondystrophic or mdx preparations. Daily treatment with PDTC significantly improved the RP regardless of age. These results are consistent with the hypothesis that passive stretch activates an NFkappaB-mediated pathogenic mechanism in dystrophic muscle and suggest that agents which stabilize cytosolic IkappaB-alpha levels may be useful for treating Duchenne and related muscular dystrophies.

Spontaneous muscle action potentials fail to develop without fetal-type acetylcholine receptors

In mammals, two combinations of muscle nicotinic acetylcholine receptors (AChRs) are used: α2βγδ (γ‐AChR) or α2βϵδ (ϵ‐AChR). After birth, γ‐AChRs are replaced by ϵ‐AChRs (γ/ϵ‐switch). The two receptors have different conductances and open times. During perinatal period, the long open time γ‐AChRs generate random myofiber action potentials from uniquantal miniature end‐plate potentials (mEPPs). ϵ‐AChRs are suitable for strong adult muscle activities. Since the effect of the γ/ϵ‐switch on neuromuscular development was unclear, despite the many differences in channel characteristics, we carried out this study to generate γ‐subunit‐deficient mice. Homozygotes born alive survived for 2 days in a stable condition, and were able to move their forelimbs. Endplate AChRs included ϵ‐subunits, and muscle fibers had multiple neuromuscular junctions. Both pre‐ and postsynapses were abnormal and spontaneous action potentials generated from mEPPs were totally absent. Results suggest a requirement for γ‐AChRs in mediating synaptically‐induced action potential activity critical for neuromuscular development.

Treatment with inhibitors of the NF-κB pathway improves whole body tension development in the mdx mouse

The whole body tension (WBT) method was used to evaluate the hypothesis that long term treatment with NF-kappaB inhibitors improves the total forward pulling tension exerted by the limb musculature of the mdx mouse. Mdx mice exhibited significantly reduced WBT values and more profound weakening during the course of generating multiple forward pulling movements than age-matched nondystrophic mice. Long term treatment with the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) did not significantly reduce nuclear p65 activation in the costal diaphragm, but increased WBT by 12% in mature (12 month) mice. Daily treatment (30 days) of 1 month old mdx mice with the inhibitor ursodeoxycholic acid (UDCA) reduced costal diaphragm nuclear p65 activation by 40% and increased WBT by 21%. These results indicate that treatment with NF-kappaB inhibitors improves WBT in the mdx mouse and further establishes the utility of the WBT procedure in assessing therapeutic efficacy.

Soluble activin receptor type IIB increases forward pulling tension in the mdx mouse

Introduction: In this study we investigated the action of RAP‐031, a soluble activin receptor type IIB (ActRIIB) comprised of a form of the ActRIIB extracellular domain linked to a murine Fc, and the NF‐κB inhibitor, ursodeoxycholic acid (UDCA), on the whole body strength of mdx mice. Methods: The whole body tension (WBT) method of assessing the forward pulling tension (FPT) exerted by dystrophic (mdx) mice was used. Results: RAP‐031 produced a 41% increase in body mass and a 42.5% increase in FPT without altering the FPT normalized for body mass (WBT). Coadministration of RAP‐031 with UDCA produced increases in FPT that were associated with an increase in WBT. Conclusions: Myostatin inhibition increases muscle mass without altering the fundamental weakness characteristic of dystrophic muscle. Cotreatment with an NF‐κB inhibitor potentiates the effects of myostatin inhibition in improving FPT in mdx mice. Muscle Nerve, 2011

A simple protocol for assessing inter-trial and inter-examiner reliability for two noninvasive measures of limb muscle strength

Noninvasive measures of limb muscle strength are quite useful in preclinical translational studies that use mouse models of muscle disease, peripheral nerve disease, and movement disorders. The present study uses a simple protocol for assessing both inter-trial and inter-examiner reliability for two noninvasive methods of assessing limb strength in dystrophic (mdx) and wild type mice. One method, termed the whole body tension (WBT) method or escape test, measures the total phasic pulling tension exerted by the fore- and hindlimbs while a mouse attempts to escape into a darkened tube. Another procedure, termed the four limb wire grid holding test, measures the minimal amount of sustained tension (physical impulse) exerted by the fore- and hindlimbs while the mouse hangs suspended in an upside-down position. A comparison of the two methods revealed significant inter-trial and inter-examiner correlations in each procedure, although the WBT procedure consistently produced higher correlations than the four limb wire grid holding test. Inter-trial reliability for each test was higher than inter-examiner reliability, indicating that each longitudinal series of tests is best performed by a single investigator. The holding test also did not consistently detect differences between wild type and mdx populations at ages greater than 4 months. These results demonstrate the utility of a simple protocol for assessing the reliability of noninvasive tests that measure limb strength, and should be useful in comparing different functional measures in a broad range of translational studies.

Single channel evidence for a cytoskeletal defect involving acetylcholine receptors and calcium influx in cultured dystrophic (MDX) myotubes

Single channel events that exhibited the conductance, event duration, and ion selectivity characteristics of calcium leakage activity (CLA) were recorded in association with acetylcholine receptor (AChR) activity in cultured nondystrophic myotubes. The CLA was observed in the presence or absence of acetylcholine (ACh), and at normal or elevated concentrations of calcium. In contrast to results from nondystrophic myotubes, cell‐attached patches from several cultured dystrophic (mdx) myotubes exhibited 100% CLA with no AChR activity, even though ACh was present in the pipette solution. Acquisition of an inside‐out patch from these membrane areas produced a profound decrease in CLA and the appearance of AChR events exhibiting typical conductance and event duration characteristics. These results suggest that CLA in dystrophic muscle is produced, in part, by unusual physical interactions between AChRs and the dystrophic cytoskeleton that are mediated by the action of intracellular modulators responsible for aggregating and stabilizing AChRs.

Increases in nuclear p65 activation in dystrophic skeletal muscle are secondary to increases in the cellular expression of p65 and are not solely produced by increases in IκB-α kinase activity

Dystrophin-deficient muscle exhibits substantial increases in nuclear NF-kappaB activation. To examine potential mechanisms for this enhanced activation, the present study employs conventional Western blot techniques to provide the first determination of the relative expression of NF-kappaB signaling molecules in adult nondystrophic and dystrophic (mdx) skeletal muscle. The results indicate that dystrophic muscle is characterized by increases in the whole cell expression of IkappaB-alpha, p65, p50, RelB, p100, p52, IKK, and TRAF-3. The proportion of phosphorylated IkappaB-alpha, p65, NIK, and IKKbeta, and the level of cytosolic IkappaB-alpha, were also increased in the mdx diaphragm. Proteasomal inhibition using MG-132 increased the proportion of phosphorylated IkappaB-alpha in nondystrophic diaphragm, but did not significantly increase this proportion in the mdx diaphragm. This result suggests that phosphorylated IkappaB-alpha accumulates in dystrophic cytosol because the rate of IkappaB-alpha degradation is lower than the effective rate of IkappaB-alpha synthesis and phosphorylation. Dystrophic increases in SUMO-1 (small ubiquitin modifier-1) protein and in Akt activation were also observed. The results indicate that increases in nuclear p65 activation in dystrophic muscle are not produced solely by increases in the activity of IkappaB-alpha kinase (IKK), but are due primarily to increases in the expression of p65 and other NF-kappaB signaling components.

Extrajunctional resting Ca2+ influx is not increased in a severely dystrophic expiratory muscle (triangularis sterni) of the mdx mouse

Freshly isolated adult mdx and nondystrophic (C57B110SnJ) muscle fibers were used to examine the potential role of resting Ca2+ influx in the pathogenesis of Duchenne and related dystrophies. Microfluorimetric determinations of resting divalent cation influx were obtained from undissociated intact muscle fibers in the triangularis sterni (TS), a thin expiratory muscle. Morphological evidence indicated severe dystrophic alterations in the mdx TS at 5 months, and a pronounced loss of fibers with connective tissue infiltration in older animals. To examine resting Ca2+ influx, fibers were loaded with FURA PE3 and the rate of quenching of intracellular signal following the extracellular addition of Mn2+ was determined from extrajunctional regions. There was no significant difference in quench rate between nondystrophic and mdx TS fibers. These results indicate that severe dystrophic pathology in the absence of dystrophin is not due to generalized increases in resting Ca2+ influx.

Acetylcholine receptor and calcium leakage activity in nondystrophic and dystrophic myotubes (MDX)

To determine whether the lack of dystrophin alters the occurrence of calcium leakage activity (CLA) and acetylcholine receptor (AChR) activity, the frequency of each event class was determined from several cell attached patches on nondystrophic and dystrophic (mdx) myotubes. The frequency of CLA observed in the presence of ACh was significantly (P < 0.05) elevated in mdx myotubes, an effect which was partly due to a significant (P < 0.05) increase in the proportion of cell attached patches that exhibited 100% CLA with no AChR activity. Areas of mdx and nondystrophic membrane that exhibited reduced or absent AChR activity had significantly (P < 0.01) and substantially elevated calcium leakage event frequencies. This inverse and discontinuous relationship between CLA and AChR activity provides further evidence that some CLA in dystrophic muscle is produced by clusters of AChRs that form unusual physical associations with the dystrophic cytoskeleton during the processes associated with receptor localization and stabilization.

The relationship between the resistance of a membrane patch and predicted changes in total patch circuit resistance secondary to spontaneous or induced alterations in patch geometry.

The objective of this study was to determine the relationship between the magnitude of the membrane resistance in the free area of a cell-attached patch-clamp recording and the change in total patch circuit resistance that would be produced by the introduction of a series resistance, such as would be observed upon acquiring a patch-clamped membrane vesicle. The results describe a method for determining the magnitude of the membrane resistance in the free area of a membrane patch, and demonstrate that: (a) at a given value of shunt resistance, areas of membrane with higher resistivity produce smaller proportional increases in total patch circuit resistance upon acquiring a membrane vesicle; and (b) a presumption of spherical vesicle formation provides a lower limit estimate of the membrane resistance. The described procedures and relationships are useful in developing new techniques for examining channel activity in membrane patches where individual events are below the present limits of detection, for examining changes in membrane resistivity and/or shunt resistance in patches undergoing cytoskeletal re-organization, and for assessing the potential influence of series resistance changes on single channel parameters in longer term cell-attached patch-clamp recordings.