Transforming Growth Factor Beta (TGF-β) Is a Muscle Biomarker of Disease Progression in ALS and Correlates with Smad Expression

We recently identified Smads1, 5 and 8 as muscle biomarkers in human ALS. In the ALS mouse, these markers are elevated and track disease progression. Smads are signal transducers and become activated upon receptor engagement of ligands from the TGF-β superfamily. Here, we sought to characterize ligands linked to activation of Smads in ALS muscle and their role as biomarkers of disease progression. RNA sequencing data of ALS muscle samples were mined for TGF-β superfamily ligands. Candidate targets were validated by qRT-PCR in a large cohort of human ALS muscle biopsy samples and in the G93A SOD1 mouse. Protein expression was evaluated by Western blot, ELISA and immunohistochemistry. C2C12 muscle cells were used to assess Smad activation and induction. TGF-β1, 2 and 3 mRNAs were increased in ALS muscle samples compared to controls and correlated with muscle strength and Smads1, 2, 5 and 8. In the G93A SOD1 mouse, the temporal pattern of TGF-β expression paralleled the Smads and increased with disease progression. TGF-β1 immunoreactivity was detected in mononuclear cells surrounding muscle fibers in ALS samples. In muscle cells, TGF-β ligands were capable of activating Smads. In conclusion, TGF-β1, 2 and 3 are novel biomarkers of ALS in skeletal muscle. Their correlation with weakness in human ALS and their progressive increase with advancing disease in the ALS mouse suggest that they, as with the Smads, can track disease progression. These ligands are capable of upregulating and activating Smads and thus may contribute to the Smad signaling pathway in ALS muscle.     

Myogenic protein expression before and after resistance loading in 26- and 64-yr-old men and women.

Based on the growing body of evidence implicating an important role for myogenic regulatory factors (MRFs) in the adaptive responses of skeletal muscle to mechanical load, we tested the hypothesis that protein concentrations of MRFs as well as cell cycle proteins (i.e., cyclins and cyclin-dependent kinase inhibitors) would be altered after heavy leg resistance exercise (RE). Because we and others, however, have shown a blunted adaptive response to long-term resistance training in older (O) women [females (F)] compared with men (M), we also tested the hypothesis that these myogenic responses to RE would be influenced by age and gender. Twenty-two younger (Y) adults (20-35 yr, 11 YF, 11 YM) and 20 O adults (60-75 yr, 9 OF, 11 OM) consented to vastus lateralis muscle biopsy before and 24 h after a bout of RE using a regimen known to induce myofiber hypertrophy when performed 2-3 days/wk for several weeks (3 sets of 80% one-repetition maximum for squat, leg press, and knee extension). Protein concentrations of MRFs (MyoD, myogenin, myf-6), cyclin D1, cyclin B1, alpha-actin, and the cyclin-dependent kinase inhibitor p27kip were determined by immunoblotting. Data were analyzed by using age x gender x load repeated-measures ANOVA. Myogenin expression was 44% higher (P <0.05) in O compared with Y, and myf-6 tended to be higher in OF compared with YF (95%, P=0.059). A significant gender x load interaction indicated that, in F, RE led to a reduction in p27kip (20%; P<0.05), which was driven mainly by a 27% drop in OF. Levels of cyclin D1, cyclin B1, MyoD, myf-6, and alpha-actin were not influenced by age, gender, or loading. We report a novel finding in humans of markedly higher myogenin protein content in older sedentary muscle. The results do not, however, support the hypothesis that myogenic protein expression is altered 24 h after RE, irrespective of age or gender. Although the time point of postexercise muscle biopsy could be viewed as too early to capture maximal effects for most of these proteins, the significant decline in p27kip concentration found in OF suggests that mechanical load may provide one means of overcoming the inhibitory influence of p27kip.     

Age is independently related to muscle metabolic capacity in premenopausal women.

The purpose of this study was to determine whether muscle metabolic capacity was inversely related to age after adjusting for physical activity in sedentary premenopausal women. Eighty-three women (ages 23-47 yr) had their free-living, activity-related energy expenditure evaluated with doubly labeled water procedures, and room calorimeter determined sleeping energy expenditure. Maximum O(2) uptake and strength were evaluated in all subjects, whereas 31P-magnetic resonance spectroscopy determined metabolic economy during maximal exercise, and muscle biopsy maximal enzyme activity was evaluated in subsets of the sample (48 and 18 subjects, respectively). Age was significantly related to whole body treadmill endurance time (r = -0.32), plantar flexion strength (r = -0.29), maximum O(2) uptake (r = -0.27), (31)P-magnetic resonance spectroscopy ADP recovery rate (r = -0.44), and anaerobic glycolytic capacity (r = -0.37), and muscle biopsy citrate synthase activity (r = -0.48), glyceraldehyde-3-phosphate dehydrogenase (r = -0.54), phosphofructokinase (r = -0.62), and phosphorylase (r = -0.58) activity even after adjusting for activity-related energy expenditure. These data suggest that, in sedentary premenopausal women, both oxidative and glycolytic muscle capacity decrease with age even when physical activity is taken into account.     

Age differences in knee extension power, contractile velocity, and fatigability.

The purposes of this study were to examine age and gender differences in knee extensor strength, power, and fatigue using open- and closed-chain testing procedures. We tested the hypothesis that specific strength (strength/unit muscle mass) would not differ by age, whereas age differences in specific power and fatigue would remain consequent to blunted maximal contractile velocity. Skeletal muscle performance was examined in 28 young (26.9 +/- 0.7 yr) and 24 older (63.6 +/- 0.8 yr) men and women. Assessments included one-repetition maximum strength for knee extension, leg press, and squat; concentric knee extensor peak power, velocity, and fatigability; and sit-to-stand power, fatigability, and relative neural activation (electromyograph activity during sit-to-stand movement normalized to electromyograph activity during isometric maximum voluntary contraction). Thigh lean mass (TLM; kg) was assessed by dual-energy X-ray absorptiometry. Specific strength (N/kg TLM) and specific power (W/kg TLM) were estimated by dividing absolute values by TLM. Age differences in specific strength were observed for knee extension only (young, 41.2 +/- 1.0 N/kg TLM; older, 32.4 +/- 1.0 N/kg TLM; P < 0.05). Adjustment for TLM did not negate age differences in knee extension specific power (25-41% lower in older; P < 0.05) across loads tested. Older adults experienced fatigue across 10 repetitions of knee extension as peak velocity fell by 24% (P < 0.05). Deficits in concentric power persist after adjustment for TLM as maximum contractile velocity falls markedly with aging. Older adults are less capable of sustaining maximum concentric velocity during repetitive contractions. These findings suggest that velocity impairments are a possible contributor to mobility loss and falls risk among older adults. Interventions for improving contractile velocity should be pursued.     

Ethnic differences in triceps surae muscle-tendon complex and walking economy

The purposes of this study were to (a) determine whether structural differences in triceps surae muscle-tendon complex and walking economy exist between 14 African American and 19 Caucasian sedentary women and (b) determine whether muscle-tendon parameters are associated with walking economy. African American and Caucasian subjects were matched on body weight, height, and body composition. Muscle-tendon parameters were determined by magnetic resonance imaging and walking economy was evaluated at 4.8 km.h(-1). Medial gastrocnemius and total triceps surae muscle shape were different across ethnicity despite no ethnic differences in plantar flexion strength or in maximal cross-sectional area for any triceps surae muscles. African American women had shorter gastrocnemius muscles and longer tendons and performed walking more economically. Tendon length was the only variable related to walking economy. No ethnic differences were observed in walking economy after adjusting for tendon length. Data show gastrocnemius tendon length is related to level walking and longer gastrocnemius tendons may partly explain more economical walking in African American women. These preliminary findings indicate the structure of the muscle-tendon complex could be a factor partially accounting for reported ethnic differences in certain types of athletic-related performance.     

Bed rest decreases mechanically induced myofiber wounding and consequent wound-mediated FGF release

Using a terrestrial model of spaceflight (i.e., bed rest), weinvestigated the amount of myofiber wounding and fibroblast growthfactor (FGF) release that occurs during unloading.Myofiber wounding was determined by serum levels of the creatine kinase MM (CKMM) isoform before and after bed rest. Serum levels of both acidic FGF (aFGF) and basic FGF were also determined. A second group ofsubjects was treated in an identical fashion except that they underwenta resistive exercise program during bed rest. Bed rest alone causedsignificant (P < 0.05;n = 7) reductions in post-bed-restserum levels of both CKMM and aFGF, which were paralleled by asignificant (P < 0.05;n = 7) decrease in myofiber size. Incontrast, bed rest plus resistive exercise resulted in significant (P < 0.05;n = 7) increases in post-bed-restserum levels of both CKMM and aFGF, which were paralleled by inhibitionof the atrophic response. These results suggest that mechanicallyinduced, myofiber wound-mediated FGF release may play an important rolein the etiology of unloading-induced skeletal muscle atrophy.

     

Resistance exercise training and the orthostatic response

Resistance exercise has been suggested to increase blood volume, increase the sensitivity of the carotid baroreceptor cardiac reflex response (BARO), and decrease leg compliance, all factors that are expected to improve orthostatic tolerance. To further test these hypotheses, cardiovascular responses to standing and to pre-syncopal limited lower body negative pressure (LBNP) were measured in two groups of sedentary men before and after a 12-week period of either exercise (n = 10) or no exercise (control, n = 9). Resistance exercise training consisted of nine isotonic exercises, four sets of each, 3 days per week, stressing all major muscle groups. After exercise training, leg muscle volumes increased (P < 0.05) by 4–14%, lean body mass increased (P = 0.00) by 2.0 (0.5) kg, leg compliance and BARO were not significantly altered, and the maximal LBNP tolerated without pre-syncope was not significantly different. Supine resting heart rate was reduced (P = 0.03) without attenuating the heart rate or blood pressure responses during the stand test or LBNP. Also, blood volume (¹²⁵I and ⁵¹Cr) and red cell mass were increased (P < 0.02) by 2.8% and 3.9%, respectively. These findings indicate that intense resistance exercise increases blood volume but does not consistently improve orthostatic tolerance. Accepted: 17 January 1997