Ted G. Graber

Clinically Relevant Frailty Index for Mice

Frailty is a clinical syndrome associated with the aging process and adverse outcomes. The purpose of this short report was to initiate the development of a Frailty Index in 27- to 28-month-old C57BL/6 mice that matched the clinical criteria used in humans (weakness, slow walking speed, low activity level, poor endurance). The selected criteria included grip strength, walking speed, physical activity, and endurance. The criteria in mice were evaluated by the inverted-cling grip test, rotarod test, voluntary wheel running, and derived endurance scores. Each criterion had a designated cutoff point (1.5 SD below the cohort mean) to identify the mice with the lowest performance. If a mouse presented with three of the criteria scores below the cutoff points, it was identified as frail. Mild frailty was designated if two criteria were below the cutoff points. In this mouse cohort, one mouse was identified as frail and one was mildly frail. This prevalence of 9% frailty is consistent with the prevalence of frailty in humans at the same survival age. Collectively, our selected criterion, cutoff point, and Frailty Index provide a potential standardized definition for frailty in mice that is consistent with the operational definition of frailty in humans.

C57BL/6 Neuromuscular Healthspan Scoring System

Developing a scoring system based on physiological and functional measurements is critical to test the efficacy of potential interventions for sarcopenia and frailty in aging animal models; therefore, the aim of this study was to develop a neuromuscular healthspan scoring system (NMHSS). We examined three ages of male C57BL/6 mice: adults (6-7 months old, 100% survival), old (24-26 months old, 75% survival), and elderly group (>28 months old, ≤50% survival)-as well as mice along this age continuum. Functional performance (as determined by the rotarod and inverted-cling grip test) and in vitro muscle contractility were the determinants. A raw score was derived for each determinant, and the NMHSS was then derived as the sum of the individual determinant scores. In comparison with individual determinants, the NMHSS reduced the effect of individual variability within age groups, thus potentially providing an enhanced ability to detect treatment effects in future studies.

Voluntary Aerobic Exercise Reverses Frailty in Old Mice

Frailty is a major cause of disability and loss of independence in the elderly. Using clinically relevant criteria from our previously established mouse frailty index, we investigated the effects of aerobic exercise on frailty in male C57BL/6 mice. In order to measure the effect of treatment on the individual animals, we constructed a composite score, the Frailty Intervention Assessment Value. We hypothesized voluntary aerobic exercise would improve individual criteria and reverse or prevent frailty in the old mice. Five adult and 11 old mice (6 and 28+ months, respectively) were housed individually in cages with running wheels for 4 weeks. Controls (adult, n = 5 and old, n = 17) were housed without wheels. Inverted cling grip and rotarod tests were performed pre- and postintervention. Hind limb muscles were used for biochemical analysis and contractility experiments. We conclude that the exercise stimulus reversed frailty and was sufficient to maintain or improve functional performance in old mice, as well as to produce measurable morphological changes. In addition, the Frailty Intervention Assessment Value proved to be a valuable tool with increased power to detect treatment effects and to examine the intervention efficacy at the level of the individual mouse.

Immunoproteasome in animal models of Duchenne muscular dystrophy

Abstract Increased proteasome activity has been implicated in the atrophy and deterioration associated with dystrophic muscles of Duchenne muscular dystrophy (DMD). While proteasome inhibitors show promise in the attenuation of muscle degeneration, proteasome inhibition-induced toxicity was a major drawback of this therapeutic strategy. Inhibitors that selectively target the proteasome subtype that is responsible for the loss in muscle mass and quality would reduce side effects and be less toxic. This study examined proteasome activity and subtype populations, along with muscle function, morphology and damage in wild-type (WT) mice and two murine models of DMD, dystrophin-deficient (MDX) and dystrophin- and utrophin-double-knockout (DKO) mice. We found that immunoproteasome content was increased in dystrophic muscles while the total proteasome content was unchanged among the three genotypes of mice. Proteasome proteolytic activity was elevated in dystrophic muscles, especially in DKO mice. These mice also exhibited more severe muscle atrophy than either WT or MDX mice. Muscle damage and regeneration, characterized by the activity of muscle creatine kinase in the blood and the percentage of central nuclei were equally increased in dystrophic mice. Accordingly, the overall muscle function was similarly reduced in both dystrophic mice compared with WT. These data demonstrated that there was transformation of standard proteasomes to immunoproteasomes in dystrophic muscles. In addition, DKO that showed greatest increase in proteasome activities also demonstrated more severe atrophy compared with MDX and WT. These results suggest a putative role for the immunoproteasome in muscle deterioration associated with DMD and provide a potential target for therapeutic intervention.

Repetitive TLR3 activation in the lung induces skeletal muscle adaptations and cachexia

&NA; Due to immunosenescence, older adults are particularly susceptible to lung‐based viral infections, with increased severity of symptoms in those with underlying chronic lung disease. Repeated respiratory viral infections produce lung maladaptations, accelerating pulmonary dysfunction. Toll like 3 receptor (TLR3) is a membrane protein that senses exogenous double‐stranded RNA to activate the innate immune response to a viral infection. Polyinosinic‐polycytidylic acid [poly(I:C)] mimics double stranded RNA and has been shown to activate TLR3. Utilizing an established mouse viral exacerbation model produced by repetitive intranasal poly(I:C) administration, we sought to determine whether repetitive poly(I:C) treatment induced negative muscle adaptations (i.e. atrophy, weakness, and loss of function). We determined skeletal muscle morphological properties (e.g. fiber‐type, fiber cross‐sectional area, muscle wet mass, etc.) from a treated group ((poly(I:C), n = 9) and a sham‐treated control group (PBS, n = 9); age approximately 5 months. In a subset (n = 4 for both groups), we determined in vivo physical function (using grip test for strength, rotarod for overall motor function, and treadmill for endurance) and muscle contractile properties with in vitro physiology (in the EDL, soleus and diaphragm). Our findings demonstrate that poly(I:C)‐treated mice exhibit both muscle morphological and functional deficits. Changes of note when comparing poly(I:C)‐treated mice to PBS‐treated controls include reductions in fiber cross‐sectional area (−27% gastrocnemius, −25% soleus, −16% diaphragm), contractile dysfunction (soleus peak tetanic force, −26%), muscle mass (gastrocnemius −19%, soleus −23%), physical function (grip test −34%), body mass (−20%), and altered oxidative capacity (140% increase in succinate dehydrogenase activity in the diaphragm, but 66% lower in the gastrocnemius). Our data is supportive of a new model of cachexia/sarcopenia that has potential for future research into the mechanisms underlying muscle wasting.