Barbara K. Smith

Inspiratory muscle strength training improves weaning outcome in failure to wean patients: a randomized trial

IntroductionMost patients are readily liberated from mechanical ventilation (MV) support, however, 10% - 15% of patients experience failure to wean (FTW). FTW patients account for approximately 40% of all MV days and have significantly worse clinical outcomes. MV induced inspiratory muscle weakness has been implicated as a contributor to FTW and recent work has documented inspiratory muscle weakness in humans supported with MV.MethodsWe conducted a single center, single-blind, randomized controlled trial to test whether inspiratory muscle strength training (IMST) would improve weaning outcome in FTW patients. Of 129 patients evaluated for participation, 69 were enrolled and studied. 35 subjects were randomly assigned to the IMST condition and 34 to the SHAM treatment. IMST was performed with a threshold inspiratory device, set at the highest pressure tolerated and progressed daily. SHAM training provided a constant, low inspiratory pressure load. Subjects completed 4 sets of 6-10 training breaths, 5 days per week. Subjects also performed progressively longer breathing trials daily per protocol. The weaning criterion was 72 consecutive hours without MV support. Subjects were blinded to group assignment, and were treated until weaned or 28 days.ResultsGroups were comparable on demographic and clinical variables at baseline. The IMST and SHAM groups respectively received 41.9 ± 25.5 vs. 47.3 ± 33.0 days of MV support prior to starting intervention, P = 0.36. The IMST and SHAM groups participated in 9.7 ± 4.0 and 11.0 ± 4.8 training sessions, respectively, P = 0.09. The SHAM groups pre to post-training maximal inspiratory pressure (MIP) change was not significant (-43.5 ± 17.8 vs. -45.1 ± 19.5 cm H2O, P = 0.39), while the IMST groups MIP increased (-44.4 ± 18.4 vs. -54.1 ± 17.8 cm H2O, P < 0.0001). There were no adverse events observed during IMST or SHAM treatments. Twenty-five of 35 IMST subjects weaned (71%, 95% confidence interval (CI) = 55% to 84%), while 16 of 34 (47%, 95% CI = 31% to 63%) SHAM subjects weaned, P = .039. The number of patients needed to be treated for effect was 4 (95% CI = 2 to 80).ConclusionsAn IMST program can lead to increased MIP and improved weaning outcome in FTW patients compared to SHAM treatment.Trial RegistrationClinicalTrials.gov: NCT00419458

Pompe disease gene therapy

Pompe disease is an autosomal recessive metabolic myopathy caused by the deficiency of the lysosomal enzyme acid alpha-glucosidase and results in cellular lysosomal and cytoplasmic glycogen accumulation. A wide spectrum of disease exists from hypotonia and severe cardiac hypertrophy in the first few months of life due to severe mutations to a milder form with the onset of symptoms in adulthood. In either condition, the involvement of several systems leads to progressive weakness and disability. In early-onset severe cases, the natural history is characteristically cardiorespiratory failure and death in the first year of life. Since the advent of enzyme replacement therapy (ERT), the clinical outcomes have improved. However, it has become apparent that a new natural history is being defined in which some patients have substantial improvement following ERT, while others develop chronic disability reminiscent of the late-onset disease. In order to improve on the current clinical outcomes in Pompe patients with diminished clinical response to ERT, we sought to address the cause and potential for the treatment of disease manifestations which are not amenable to ERT. In this review, we will focus on the preclinical studies that are relevant to the development of a gene therapy strategy for Pompe disease, and have led to the first clinical trial of recombinant adeno-associated virus-mediated gene-based therapy for Pompe disease. We will cover the preliminary laboratory studies and rationale for a clinical trial, which is based on the treatment of the high rate of respiratory failure in the early-onset patients receiving ERT.

The respiratory neuromuscular system in Pompe disease.

Pompe disease is due to mutations in the gene encoding the lysosomal enzyme acid α-glucosidase (GAA). Absence of functional GAA typically results in cardiorespiratory failure in the first year; reduced GAA activity is associated with progressive respiratory failure later in life. While skeletal muscle pathology contributes to respiratory insufficiency in Pompe disease, emerging evidence indicates that respiratory neuron dysfunction is also a significant part of dysfunction in motor units. Animal models show profound glycogen accumulation in spinal and medullary respiratory neurons and altered neural activity. Tissues from Pompe patients show central nervous system glycogen accumulation and motoneuron pathology. A neural mechanism raises considerations about the current clinical approach of enzyme replacement since the recombinant protein does not cross the blood-brain-barrier. Indeed, clinical data suggest that enzyme replacement therapy delays symptom progression, but many patients eventually require ventilatory assistance, especially during sleep. We propose that treatments which restore GAA activity to respiratory muscles, neurons and networks will be required to fully correct ventilatory insufficiency in Pompe disease.

B-cell depletion is protective against anti-AAV capsid immune response: a human subject case study

Gene therapy strategies for congenital myopathies may require repeat administration of adeno-associated viral (AAV) vectors due to aspects of the clinical application, such as: (i) administration of doses below therapeutic efficacy in patients enrolled in early phase clinical trials; (ii) progressive reduction of the therapeutic gene expression over time as a result of increasing muscle mass in patients treated at a young age; and (iii) a possibly faster depletion of pathogenic myofibers in this patient population. Immune response triggered by the first vector administration, and to subsequent doses, represents a major obstacle for successful gene transfer in young patients. Anti-capsid and anti-transgene product related humoral and cell-mediated responses have been previously observed in all preclinical models and human subjects who received gene therapy or enzyme replacement therapy (ERT) for congenital myopathies. Immune responses may result in reduced efficacy of the gene transfer over time and/or may preclude for the possibility of re-administration of the same vector. In this study, we evaluated the immune response of a Pompe patient dosed with an AAV1-GAA vector after receiving Rituximab and Sirolimus to modulate reactions against ERT. A key finding of this single subject case report is the observation that B-cell ablation with rituximab prior to AAV vector exposure results in non-responsiveness to both capsid and transgene, therefore allowing the possibility of repeat administration in the future. This observation is significant for future gene therapy studies and establishes a clinically relevant approach to blocking immune responses to AAV vectors.

Establishing Clinical End Points of Respiratory Function in Large Animals for Clinical Translation

Respiratory dysfunction due progressive weakness of the respiratory muscles, particularly the diaphragm, is a major cause of death in the neuromuscular disease (NMD) X-linked myotubular myopathy (XLMTM). Methods of respiratory assessment in patients are often difficult, especially in those who are mechanically ventilated. The naturally occuring XLMTM dog model exhibits a phenotype similar to that in patients and can be used to determine quantitative descriptions of dysfunction as clinical endpoints for treatment and the development of new therapies. In experiments using respiratory impedance plethysmography (RIP), XLMTM dogs challenged with the respiratory stimulant doxapram displayed significant changes indicative of diaphragmatic weakness.

Transcriptional regulation of myotrophic actions by testosterone and trenbolone on androgen-responsive muscle

Androgens regulate body composition and skeletal muscle mass in males, but the molecular mechanisms are not fully understood. Recently, we demonstrated that trenbolone (a potent synthetic testosterone analogue that is not a substrate for 5-alpha reductase or for aromatase) induces myotrophic effects in skeletal muscle without causing prostate enlargement, which is in contrast to the known prostate enlarging effects of testosterone. These previous results suggest that the 5α-reduction of testosterone is not required for myotrophic action. We now report differential gene expression in response to testosterone versus trenbolone in the highly androgen-sensitive levator ani/bulbocavernosus (LABC) muscle complex of the adult rat after 6weeks of orchiectomy (ORX), using real time PCR. The ORX-induced expression of atrogenes (Muscle RING-finger protein-1 [MuRF1] and atrogin-1) was suppressed by both androgens, with trenbolone producing a greater suppression of atrogin-1 mRNA compared to testosterone. Both androgens elevated expression of anabolic genes (insulin-like growth factor-1 and mechano-growth factor) after ORX. ORX-induced increases in expression of glucocorticoid receptor (GR) mRNA were suppressed by trenbolone treatment, but not testosterone. In ORX animals, testosterone promoted WNT1-inducible-signaling pathway protein 2 (WISP-2) gene expression while trenbolone did not. Testosterone and trenbolone equally enhanced muscle regeneration as shown by increases in LABC mass and in protein expression of embryonic myosin by western blotting. In addition, testosterone increased WISP-2 protein levels. Together, these findings identify specific mechanisms by which testosterone and trenbolone may regulate skeletal muscle maintenance and growth.

Phrenic Nerve Stimulation Increases Human Diaphragm Fiber Force after Cardiothoracic Surgery

To the Editor: Mechanical ventilation (MV) causes diaphragm inactivity and unloading, and this quiescence results in ventilator-induced diaphragm dysfunction (VIDD). VIDD is characterized by oxidative stress, muscle atrophy, and decreases in diaphragm fiber force, as documented in both animal models and humans (1). VIDD is a major clinical problem because the ensuing decreased ability to develop inspiratory pressure will contribute to the difficulties in weaning patients from MV. During open-chest cardiothoracic surgery in humans, a rapid form of VIDD has been observed, with 20–30% depression of diaphragm force occurring within 2 hours of commencing MV (2). Extensive experiments establishing a cause-and-effect relationship for the mechanisms leading to VIDD in animals are available (reviewed in Reference 3), but interventions in humans are scant. Recently, intermittent phrenic nerve stimulation during MV has emerged as a potential VIDD countermeasure (4, 5). Specifically, hemidiaphragm stimulation during MV resulted in greater type 2 muscle fiber area in sheep (4), improved contractile properties in rats (6), and higher mitochondrial respiration rates in humans undergoing cardiothoracic surgery (5). In this preliminary study, we examined whether intermittent phrenic nerve stimulation during cardiothoracic surgery prevents diaphragm fiber contractile function impairment. We obtained diaphragm biopsies from four patients undergoing sternotomy for elective cardiothoracic surgical procedures (Table 1). The University of Florida Institutional Review Board approved the protocol, and participants gave written consent. Patients received nondepolarizing neuromuscular blockers during intubation, but none during surgery. The experimental intervention consisted of unilateral of phrenic nerve stimulation using an external cardiac pacemaker (Medtronic 5388; Medtronic, Minneapolis, MN) with temporary wire electrodes (AE Medical Corp., Farmingdale, NJ). The stimulation parameters and protocol were reported previously (5) and are given in Table 1. Briefly, stimulations were conducted every 30 minutes as soon as the phrenic nerve and diaphragm were exposed. The contralateral hemidiaphragm served as intrasubject control. There were no complications from the stimulations or biopsies. All participants were successfully extubated on the first attempt. Table 1. Patient Characteristics and Stimulation Parameters Muscle biopsies from each hemidiaphragm were either frozen immediately and later prepared for protein immunoblotting (see online supplement) or placed in an ice-cold solution containing low calcium and high ATP concentration (“relaxing” solution) (7), processed for chemical permeabilization (7), and stored at −20°C in relaxing solution (50% glycerol) until measurements of contractile properties (≤3 wk of surgery). We carefully isolated single fibers from diaphragm bundles in ice-cold relaxing solution and clamped the fibers to a force transducer and length controller for mechanical testing at 15°C, using calcium activation (7). We discarded fibers with sarcomere length greater than 2.65 μm under slack conditions on final mounting on the mechanics apparatus. This was the only exclusion criterion we adopted to avoid selectively studying “healthy” fibers. In our hands, slack sarcomere length greater than 2.65 μm is a sign of damage imposed during isolation of the fiber. We studied fibers from each hemidiaphragm (stimulated or control) in all subjects and determined fiber types using myosin heavy chain gel electrophoresis (8). Incidentally, the vast majority of fibers were type 2 (38 fibers); only four fibers were type 1 (three control, one stimulated). Hence, we report here only findings for type 2 fibers. These fibers account for approximately half of the diaphragm fiber composition (2). We analyzed the data from four patients with a varying number of fibers in each of the stimulated and control muscles, using a mixed effects model with random intercept for each individual and a fixed effect for group (stimulated or control), using the parametric bootstrap to assess statistical significance, as described by Faraway (9). This statistical approach takes into consideration the within-subject design in which different numbers of fibers are analyzed per subject in each condition. Specific force averaged 77 kN/m2 in control/inactive fibers compared with 100–150 kN/m2 reported in healthy active type 2 fibers [rodents (7), humans (2)]. Stimulated fibers elicited an approximately 30% greater specific force compared with the inactive fibers (P = 0.028; Figure 1). We found no differences between stimulated and control hemidiaphragms in other measures of isometric contractile properties (Figure 1). We also measured the abundance of protein carbonyls and ubiquitin conjugates (n = 3 patients), which, respectively, are general markers of oxidative damage and targeting for degradation by the ubiquitin-proteasome system. There was no difference between stimulated and control hemidiaphragms for protein carbonyls (control, 8.5 ± 2.8; stimulated, 9.2 ± 3.1) or ubiquitin conjugates (control, 2.3 ± 1.1; stimulated, 2.5 ± 1.0; Figures E1–E2 in the online supplement). Figure 1. Phrenic nerve stimulation increases diaphragm fiber force after cardiothoracic surgery. (A) Specific force versus pCa relationship for all fibers from control and stimulated hemidiaphragms. Specific force is absolute force (in kilonewtons) normalized ... Several mechanisms are involved in VIDD, including activation of proteolytic pathways, oxidative stress, impaired excitation–contraction coupling, and posttranslational modification of sarcomeric proteins. Our preliminary findings suggest that intermittent phrenic nerve stimulation preserves human diaphragm fiber contractile function, possibly by inhibiting proteolysis and/or posttranslational modification of sarcomeric proteins seen after MV (3, 10). However, the protection does not appear to be mediated by changes in protein carbonyls or ubiquitination. The resolution of specific cellular and molecular mechanisms will require a larger number of subjects and different analysis techniques. It is possible that indirect stretch imposed onto the control hemidiaphragm by stimulation of the treated side conferred some protection against VIDD (11). Therefore, our data may underestimate the benefits of intermittent phrenic nerve stimulation to diaphragm fiber function. However, our intrasubject design was sufficiently sensitive to detect significant differences in function that may be obscured by the large variability in an intersubject design resulting from factors such as age, sex, genetics, duration of surgery, and so on. We cannot determine whether a 30% increase in diaphragm fiber force is clinically relevant. Of note, an increase in inspiratory/diaphragm muscle strength elicited by inspiratory strength training is associated with greater weaning rate than standard care in chronic failure-to-wean patients (12). Phrenic nerve stimulation has been shown to increase the type 2 fiber cross-sectional area in sheep (4), mitochondrial respiration rates (5), and specific force in rats (6) and humans, as shown for the first time to our knowledge in the present study. These observations suggest that intermittent diaphragm activity during MV offers some protection against VIDD. Thus, intermittent diaphragm activity may ultimately prove to be a useful clinical strategy to prevent or attenuate VIDD in a subset of patients that has yet to be identified in larger trials.

Gene expression changes in the human diaphragm after cardiothoracic surgery

OBJECTIVE We examined the effects of cardiothoracic surgery, including cardiopulmonary bypass and controlled mechanical ventilation, on messenger RNA gene expression in human diaphragm. We hypothesized that genes responsible for stress response, redox regulation, protein turnover, energy metabolism, and contractile function would be altered by cardiothoracic surgery. METHODS Paired diaphragm biopsy samples were obtained from 5 male patients (67 ± 11 years) during cardiothoracic surgery, the first as soon as the diaphragm was exposed and the second as late in surgery as possible (4.9 ± 1.8 hours between samples). We profiled messenger RNA from 5 specimen pairs with microarray analysis (Hu U133 plus 2.0; Affymetrix UK Ltd, High Wycombe, UK). Quantitative reverse transcriptase polymerase chain reaction was performed with a select set of genes exhibiting differential expression for validation. RESULTS Microarray analysis identified 779 differentially expressed (early vs late samples) unique gene products (P < .005). Postoperatively, genes related to stress response and redox regulation were upregulated. Additionally, we found significantly upregulated expression of cathepsin C (2.7-fold), cathepsin L1 (2.0-fold), various ubiquitin-conjugating enzymes (E2, approximately 1.8-fold), proinflammatory cytokine interleukin 6 (15.6-fold), and muscle-specific ubiquitin ligase (MuRF-1, 2.6-fold). Comparison of fold change values obtained by quantitative reverse transcriptase polymerase chain reaction and microarray yielded significant correlation (r = 0.95, P < .0001). CONCLUSIONS Cardiothoracic surgery results in rapid changes in human diaphragm gene expression in the operating room, including genes related to stress response, inflammation, redox regulation, and proteolysis. These results may provide insight into diaphragm muscle biology after prolonged cardiothoracic procedures.

Inspiratory muscle conditioning exercise and diaphragm gene therapy in Pompe disease: Clinical evidence of respiratory plasticity.

ABSTRACT Pompe disease is an inherited disorder due to a mutation in the gene that encodes acid &agr;‐glucosidase (GAA). Children with infantile‐onset Pompe disease develop progressive hypotonic weakness and cardiopulmonary insufficiency that may eventually require mechanical ventilation (MV). Our team conducted a first in human trial of diaphragmatic gene therapy (AAV1‐CMV‐GAA) to treat respiratory neural dysfunction in infantile‐onset Pompe. Subjects (aged 2–15 years, full‐time MV: n = 5, partial/no MV: n = 4) underwent a period of preoperative inspiratory muscle conditioning exercise. The change in respiratory function after exercise alone was compared to the change in function after intramuscular delivery of AAV1‐CMV‐GAA to the diaphragm with continued exercise. Since AAV‐mediated gene therapy can reach phrenic motoneurons via retrograde transduction, we hypothesized that AAV1‐CMV‐GAA would improve dynamic respiratory motor function to a greater degree than exercise alone. Dependent measures were maximal inspiratory pressure (MIP), respiratory responses to inspiratory threshold loads (load compensation: LC), and physical evidence of diaphragm activity (descent on MRI, EMG activity). Exercise alone did not change function. After AAV1‐CMV‐GAA, MIP was unchanged. Flow and volume LC responses increased after dosing (p < 0.05 to p < 0.005), but only in the subjects with partial/no MV use. Changes in LC tended to occur on or after 180 days. At Day 180, the four subjects with MRI evidence of diaphragm descent had greater maximal voluntary ventilation (p < 0.05) and tended to be younger, stronger, and use fewer hours of daily MV. In conclusion, combined AAV1‐CMV‐GAA and exercise training conferred benefits to dynamic motor function of the diaphragm. Children with a higher baseline neuromuscular function may have greater potential for functional gains. HighlightsChildren with Pompe disease and ventilatory insufficiency received AAV1‐CMG‐GAA.Exercise alone did not change respiratory muscle function in any subjects.In less‐affected children, dynamic respiratory function improved after gene therapy.

Altered activation of the diaphragm in late-onset Pompe disease

Pompe disease is an inherited neuromuscular disorder that affects respiratory function and leads to dependence on external ventilatory support. We studied the activation of the diaphragm using bilateral phrenic magnetic stimulation and hypothesized that diaphragm compound muscle action potential (CMAP) amplitude and evoked transdiaphragmatic pressure (Twitch PDI) would correlate to disease severity. Eight patients with late onset Pompe disease (LOPD, aged 14-48 years) and four healthy control subjects completed the tests. Maximal Twitch PDI responses were progressively reduced in patients with LOPD compared to control subjects (1.4-17.1cm H2O, p<0.001) and correlated to voluntary functional tests (p<0.05). Additionally, CMAP amplitude (mA) was lower in the patients who used nighttime or fulltime ventilatory support, when compared to controls and patients who used no ventilatory support (p<0.005). However, the normalized (%peak) Twitch PDI and CMAP responses were similar between patients and controls. This suggests a loss of functional phrenic motor units in patients, with normal recruitment of remaining motor units.