Growth hormone administration and exercise effects on muscle fiber type and diameter in moderately frail older people.

OBJECTIVE: Reduced muscle mass and strength are characteristic findings of growth hormone deficiency (GHD) and aging. We evaluated measures of muscle strength, muscle fiber type, and cross sectional area in response to treatment with recombinant human growth hormone (rhGH) with or without a structured resistance exercise program in frail older subjects. DESIGN: Placebo-controlled, randomized, double blind trial. SETTING: Outpatient clinical research center at an urban university-affiliated teaching hospital. PARTICIPANTS: Thirty-one consenting older subjects (mean age 71.3 +/- 4.5 years) recruited as a subset of a larger project evaluating rhGH and exercise in older people, who underwent 62 quadricep-muscle biopsies. INTERVENTION: Random assignment to a 6-month course of one of four protocols: rhGH administered subcutaneously daily at bedtime, rhGH and a structured resistance exercise program, structured resistance exercise with placebo injections, or placebo injections only. MEASUREMENTS: Muscle biopsy specimens were obtained from the vastus lateralis muscle. Isokinetic dynamometry strength tests were used to monitor individual progress and to adjust the weights used in the exercise program. Serum insulin-like growth factor-I (IGF-I) was measured and body composition was measured using a Hologic QDR 1000W dual X-ray densitometer. RESULTS: The administration of rhGH resulted in significant increase in circulating IGF-I levels in the individuals receiving rhGH treatment. Muscle strength increased significantly in both the rhGH/exercise (+55.6%, P =.0004) as well as the exercise alone (+47.8%, P =.0005) groups. There was a significant increase in the proportion of type 2 fibers between baseline and six months in the combined rhGH treated subjects versus those not receiving rhGH (P =.027). CONCLUSIONS: Our results are encouraging in that they suggest an effect of growth hormone on a specific aging-correlated deficit. IGF-I was increased by administrating rhGH and muscle strength was increased by exercise. The administration of rhGH to frail older individuals in this study resulted in significant changes in the proportions of fiber types. Whether changes in fiber cross-sectional area or absolute number occur with long-term growth hormone administration requires further study.

Space travel directly induces skeletal muscle atrophy.

Space travel causes rapid and pronounced skeletal muscle wasting in humans that reduces their long-term flight capabilities. To develop effective countermeasures, the basis of this atrophy needs to be better understood. Space travel may cause muscle atrophy indirectly by altering circulating levels of factors such as growth hormone, glucocorticoids, and anabolic steroids and/or by a direct effect on the muscle fibers themselves. To determine whether skeletal muscle cells are directly affected by space travel, tissue-cultured avian skeletal muscle cells were tissue engineered into bioartificial muscles and flown in perfusion bioreactors for 9 to 10 days aboard the Space Transportation System (STS, i.e., Space Shuttle). Significant muscle fiber atrophy occurred due to a decrease in protein synthesis rates without alterations in protein degradation. Return of the muscle cells to Earth stimulated protein synthesis rates of both muscle-specific and extracellular matrix proteins relative to ground controls. These results show for the first time that skeletal muscle fibers are directly responsive to space travel and should be a target for countermeasure development.

Organogenesis of skeletal muscle in tissue culture.

Skeletal muscle structure is regulated by many factors, including nutrition, hormones, electrical activity, and tension. The muscle cells are subjected to both passive and active mechanical forces at all stages of development, and these forces play important but poorly understood roles in regulating muscle organogenesis and growth. For example, during embryogenesis, the rapidly growing skeleton places large passive mechanical forces on the attached muscle tissue. These forces not only help to organize the proliferating mononucleated myoblasts into the oriented, multinucleated myofibers of a functional muscle, but also tightly couple the growth rate of muscle to that of bone. Postnatally, the actively contracting, innervated muscle fibers are subjected to different patterns of active and passive tensions that regulate longitudinal and cross-sectional myofiber growth. These mechanically induced organogenic processes have been difficult to study under normal tissue culture conditions, resulting in the development of numerous methods and specialized equipment to simulate the in vivo mechanical environment (1-4). These techniques have led to the engineering of bioartificial muscles (organoids), which display many of the characteristics of in vivo muscle, including parallel arrays of postmitotic fibers organized into fascicle-like structures with tendon-like ends. They are contractile, express adult isoforms of contractile proteins, perform directed work, and can be maintained in culture for long periods.

Attenuation of skeletal muscle wasting with recombinant human growth hormone secreted from a tissue-engineered bioartificial muscle.

Skeletal muscle wasting is a significant problem in elderly and debilitated patients. Growth hormone (GH) is an anabolic growth factor for skeletal muscle but is difficult to deliver in a therapeutic manner by injection owing to its in vivo instability. A novel method is presented for the sustained secretion of recombinant human GH (rhGH) from genetically modified skeletal muscle implants, which reduces host muscle wasting. Proliferating murine C2C12 skeletal myoblasts stably transduced with the rhGH gene were tissue engineered in vitro into bioartificial muscles (C2-BAMs) containing organized postmitotic myofibers secreting 3-5 microg of rhGH/day in vitro. When implanted subcutaneously into syngeneic mice, C2-BAMs delivered a sustained physiologic dose of 2.5 to 11.3 ng of rhGH per milliliter of serum. rhGH synthesized and secreted by the myofibers was in the 22-kDa monomeric form and was biologically active, based on downregulation of a GH-sensitive protein synthesized in the liver. Skeletal muscle disuse atrophy was induced in mice by hindlimb unloading, causing the fast plantaris and slow soleus muscles to atrophy by 21 to 35% ( < 0.02). This atrophy was significantly attenuated 41 to 55% (p < 0.02) in animals that received C2-BAM implants, but not in animals receiving daily injections of purified rhGH (1 mg/kg/day). These data support the concept that delivery of rhGH from BAMs may be efficacious in treating muscle-wasting disorders.

Bioreactor perfusion system for the long-term maintenance of tissue-engineered skeletal muscle organoids.

Three-dimensional skeletal muscle organ-like structures (organoids) formed in tissue culture by fusion of proliferating myoblasts into parallel networks of long, unbranched myofibers provide an in vivo-like model for examining the effects of growth factors, tension, and space flight on muscle cell growth and metabolism. To determine the feasibility of maintaining either avian or mammalian muscle organoids in a commercial perfusion bioreactor system, we measured metabolism, protein turnover. and autocrine/paracrine growth factor release rates. Medium glucose was metabolized at a constant rate in both low-serum- and serum-free media for up to 30 d. Total organoid noncollagenous protein and DNA content decreased approximately 22-28% (P < 0.05) over a 13-d period. Total protein synthesis rates could be determined accurately in the bioreactors for up to 30 h and total protein degradation rates could be measured for up to 3 wk. Special fixation and storage conditions necessary for space flight studies were validated as part of the studies. For example, the anabolic autocrine/paracrine skeletal muscle growth factors prostaglandin F2alpha (PGF2alpha) and insulin-like growth factor-1 (IGF-1) could be measured accurately in collected media fractions, even after storage at 37 degrees C for up to 10 d. In contrast, creatine kinase activity (a marker of cell damage) in collected media fractions was unreliable. These results provide initial benchmarks for long-term ex vivo studies of tissue-engineered skeletal muscle.

Increase in percutaneous muscle biopsy yield with a suction-enhancement technique.

The percutaneous muscle biopsy technique is used in clinical practice and biomedical research. We developed a new enhanced-suction technique [suction-enhancing nipples (SEN)] and compared it with techniques currently in practice by assessing biopsy yields on anesthetized pigs. We applied the enhanced-suction technique to human subjects participating in a clinical trial. In the pig, there was a mean 91% (1.9-fold) increase in the size of the samples obtained with the 4-mm needle when SEN was used and a mean 507% (fivefold) increase in sample size when the SEN was applied to the 6-mm needles. Nine passes of the 6-mm needle with SEN obtained from five consecutive human subjects yielded a mean individual sample size of 109.4 mg or 219.4 mg per needle pass when using the double-sample technique. Adequate tissue samples for histomorphometric and other analyses were obtained in all samples obtained. The percutaneous muscle biopsy performed with enhanced suction using inexpensive, readily available nipples enhances tissue yield two- to fivefold.

Mechanical stimulation of skeletal muscle increases prostaglandin F2 alpha production, cyclooxygenase activity, and cell growth by a pertussis toxin sensitive mechanism.

Repetitive mechanical stimulation of differentiated skeletal muscle in tissue culture increased the long-term production of prostaglandin F2 alpha, an anabolic stimulator of myofiber growth. Within 4 h of initiating mechanical stimulation, the enzymatic activity of cyclooxygenase (prostaglandin GH synthase [PGHS]), a regulatory enzyme in prostaglandin synthesis, was increased 82% (P < .005), and this increase was maintained for at least 24 h. Kinetic analysis of stretch-activated cyclooxygenase activity indicated a two to threefold decrease in the enzyme's Km, with little change in its Vmax. Immunocytochemical analysis of the cell cultures indicated the presence of high levels of the mitogen-inducible isoform of cyclooxygenase (PGHS-2) in the skeletal myofibers compared to the interstitial fibroblasts. While the stretch-induced increase in cyclooxygenase enzymatic activity was not inhibited by tetrodotoxin and therefore was independent of cellular electrical activity, the G protein inhibitor pertussis toxin prevented stretch-induced cyclooxygenase activation. Pertussis toxin also inhibited stretch-induced increases in PGF2 alpha production, phospholipase D activation, and cell growth. It is concluded that stretch of skeletal muscle increases muscle cell growth through a G protein-dependent process involving the activation of cyclooxygenase, an immediate early gene product.

Growth and development of bovine fetuses and neonates representing three genotypes.

Growth was examined in bovine fetuses and neonates that typically differ in mature size and postnatal developmental pattern. Pregnancies were established from matings expected to produce early (E), late (L), and intermediate (I) maturing postnatal growth patterns. Tissues were collected at 100 and 200 d of gestation and 30 d postnatal. Muscle:body weight ratios were lower at 100 and 200 d for the E maturity type than for the L maturity type (P < .05). This differs from observations of muscle:body weight ratios made at 30 d postnatal, at which time ratios for E were either greater than (triceps brachii, P < .05) or similar to those for L. Few differences due to maturity type were observed at 100 d for bone weight:body weight ratios; however, at 200 d of gestation E bone weight:body weight ratios were generally lower (P < .05) than those for L. The genotypic relationship for bone weight:body weight ratio at 30 d postnatal was similar to that observed at 200 d of gestation. Observations of organ weight:body weight ratios revealed no clear patterns due to maturity type. The genotypic relationship for total muscle DNA content was similar to that observed for muscle weight. These results indicate that fetal muscle development differs in cattle that have different postnatal growth patterns by as early as 100 d of gestation and that differences in fetal muscle growth are related to differences in muscle hyperplasia.

Mechanical stimulation of skeletal muscle cells mitigates glucocorticoid-induced decreases in prostaglandin production and prostaglandin synthase activity.

The glucocorticoid dexamethasone (Dex) induces a decline in protein synthesis and protein content in tissue cultured, avian skeletal muscle cells, and this atrophy is attenuated by repetitive mechanical stretch. Since the prostaglandin synthesis inhibitor indomethacin mitigated this stretch attenuation of muscle atrophy, the effects of Dex and mechanical stretch on prostaglandin production and prostaglandin H synthase (PGHS) activity were examined. In static cultures, 10(-8) M Dex reduced PGF2 alpha production 55-65% and PGE2 production 84-90% after 24-72 h of incubation. Repetitive 10% stretch-relaxations of non-Dex-treated cultures increased PGF2 alpha efflux 41% at 24 h and 276% at 72 h, and increased PGE2 production 51% at 24 h and 236% at 72 h. Mechanical stimulation of Dex-treated cultures increased PGF2 alpha production 162% after 24 h, returning PGF2 alpha efflux to the level of non-Dex-treated cultures. At 72 h, stretch increased PGF2 alpha efflux 65% in Dex-treated cultures. Mechanical stimulation of Dex-treated cultures also increased PGE2 production at 24 h, but not at 72 h. Dex reduced PGHS activity in the muscle cultures by 70% after 8-24 h of incubation, and mechanical stimulation of the Dex-treated cultures increased PGHS activity by 98% after 24 h. Repetitive mechanical stimulation attenuates the catabolic effects of Dex on cultured skeletal muscle cells in part by mitigating the Dex-induced declines in PGHS activity and prostaglandin production.

Glucocorticoid-induced skeletal muscle atrophy in vitro is attenuated by mechanical stimulation.

Glucocorticoids induce rapid atrophy of fast skeletal myofibers in vivo, and either weight lifting or endurance exercise reduces this atrophy by unknown mechanisms. We examined the effects of the synthetic glucocorticoid dexamethasone (Dex) on protein turnover in tissue-cultured avian fast skeletal myofibers and determined whether repetitive mechanical stretch altered the myofiber response to Dex. In static cultures after 3-5 days, 10(-8) M Dex decreased total protein content 42-74%, total protein synthesis rates 38-56%, mean myofiber diameter 35%, myosin heavy chain (MHC) content 86%, MHC synthesis rate 44%, and fibronectin synthesis rate 29%. Repetitive 10% stretch-relaxations of the cultured myofibers for 60 s every 5 min for 3-4 days prevented 52% of the Dex-induced decrease in protein content, 42% of the decrease in total protein synthesis rate, 77% of the decrease in MHC content, 42% of the decrease in MHC synthesis rate, and 67% of the decrease in fibronectin synthesis rate. This in vitro model system will complement in vivo studies in understanding the mechanism by which mechanical activity and glucocorticoids interact to regulate skeletal muscle growth.

L6 myoblast proliferation in response to muscle-derived extracts and serum from exercise-trained rats.

Serum and skeletal muscle-derived extracts (MDE) were bioassayed for their ability to promote [3H]thymidine incorporation in L6 myoblasts to determine if exercise-training increases mitogenic growth factor activity. Rats were trained by climbing a 60 cm vertical grid with progressively greater weight for 7 and 28 days. Serum from 7 day trained rats and vastus MDE from 7 and 28 day trained rats, but not rectus femoris MDE, had increased mitogenic activity compared to serum and MDE from untrained rats. These data suggest that exercise may increase mitogenic growth factors in some skeletal muscles.

Effect of the beta-agonist cimaterol on growth and composition of neonatal rats selected for large and small body size.

The effect of subcutaneous injection of the beta-agonist cimaterol on growth and body composition of neonatal rats differing in growth potential was examined. Rats that represented substrain populations of Charles River CD rats selected for either large or small body size were used. Cimaterol administration resulted in a greater reduction in body weight gain in the Large strain rats. Body growth rate declined linearly with increasing doses of cimaterol in both strains. Percent carcass fat and protein were unchanged with cimaterol treatment. Administration of cimaterol inhibited skeletal muscle growth but caused cardiac hypertrophy. These results suggest that the effectiveness of beta-agonists is influenced by animal genotype.

Heart Rate and Lactate Levels During Weight-Training Exercise in Trained and Untrained Men.

In brief: The effects of squatting exercise on heart rate and blood lactate levels were studied in five trained and five untrained men between the ages of 22 and 37. The subjects performed squats (sets of ten repetitions until exhaustion), resting for 2 1/2 minutes between sets. Total positive work was estimated with the following formula: (bar mass + body mass) X vertical displacement X repetitions. Heart rate, lactate, and rating of perceived exertion were measured immediately before exercise, after each set, and five minutes after reaching exhaustion. The trained subjects performed more total work and had higher heart rates and lactate levels at exhaustion than the untrained subjects, though heart rates and lactate levels were lower for trained subjects at a given bar mass or submaximal work load. The findings suggest that weight training may cause adaptations that result in reduced fatigue and/or enhanced recovery.