Douglas J. Mahoney

Analysis of global mRNA expression in human skeletal muscle during recovery from endurance exercise

To search for novel transcriptional pathways that are activated in skeletal muscle after endurance exercise, we used cDNA microarrays to measure global mRNA expression after an exhaustive bout of high‐intensity cycling (∼75 min). Healthy, young, sedentary males performed the cycling bout, and skeletal muscle biopsies were taken from the vastus lateralis before, and at 3 and 48 h after exercise. We examined mRNA expression in individual muscle samples from four subjects using cDNA microarrays, used repeated‐measures significance analysis of microarray (SAM) to determine statistically significant expression changes, and confirmed selected results using real‐time RT‐PCR. In total, the expression of 118 genes significantly increased 3 h postcycling and 8 decreased. At 48 h, the expression of 29 genes significantly increased and 5 decreased. Many of these are potentially important novel genes involved in exercise recovery and adaptation, including several involved in 1) metabolism and mitochondrial biogenesis (FOXO1, PPARδ, PPARγ, nuclear receptor binding protein 2, IL‐6 receptor, ribosomal protein L2, aminolevulinate δ‐synthase 2); 2) the oxidant stress response (metalothioneins 1B, 1F, 1G, 1H, 1L, 2A, 3, interferon regulatory factor 1); and 3) electrolyte transport across membranes [Na+‐K+‐ATPase (β3), SERCA3, chloride channel 4]. Others include genes involved in cell stress, proteolysis, apoptosis, growth, differentiation, and transcriptional activation, as well as all three nuclear receptor subfamily 4A family members (Nur77, Nurr1, and Nor1). This study is the first to characterize global mRNA expression during recovery from endurance exercise, and the results provide potential insight into 1) the transcriptional contributions to homeostatic recovery in human skeletal muscle after endurance exercise, and 2) the transcriptional contributions from a single bout of endurance exercise to the adaptive processes that occur after a period of endurance exercise training.

Beneficial effects of creatine, CoQ10, and lipoic acid in mitochondrial disorders

Mitochondrial disorders share common cellular consequences: (1) decreased ATP production; (2) increased reliance on alternative anaerobic energy sources; and (3) increased production of reactive oxygen species. The purpose of the present study was to determine the effect of a combination therapy (creatine monohydrate, coenzyme Q10, and lipoic acid to target the above‐mentioned cellular consequences) on several outcome variables using a randomized, double‐blind, placebo‐controlled, crossover study design in patients with mitochondrial cytopathies. Three patients had mitochondrial encephalopathy, lactic acidosis, and stroke‐like episodes (MELAS), four had mitochondrial DNA deletions (three patients with chronic progressive external ophthalmoplegia and one with Kearns–Sayre syndrome), and nine had a variety of other mitochondrial diseases not falling into the two former groups. The combination therapy resulted in lower resting plasma lactate and urinary 8‐isoprostanes, as well as attenuation of the decline in peak ankle dorsiflexion strength in all patient groups, whereas higher fat‐free mass was observed only in the MELAS group. Together, these results suggest that combination therapies targeting multiple final common pathways of mitochondrial dysfunction favorably influence surrogate markers of cellular energy dysfunction. Future studies with larger sample sizes in relatively homogeneous groups will be required to determine whether such combination therapies influence function and quality of life. Muscle Nerve, 2006

Effect of creatine and weight training on muscle creatine and performance in vegetarians.

PURPOSEnTo compare the change in muscle creatine, fiber morphology, body composition, hydration status, and exercise performance between vegetarians and nonvegetarians with 8 wk of creatine supplementation and resistance training.nnnMETHODSnEighteen VG and 24 NV subjects (19-55 yr) were randomly assigned (double blind) to four groups: VG + creatine (VGCr, N=10), VG + placebo (VGPl, N=8), NV + creatine (NVCr, N=12), and NV + placebo (NVPl, N=12). Before and at the end of the study, muscle biopsies were taken from the vastus lateralis m, body composition was assessed by DXA, and strength was assessed using 1-RM bench press and leg press. Subjects participated in the same 8-wk resistance-training program. Creatine dosage was based on lean tissue mass (0.25 g.kg(-1) LTM.d(-1) x 7 d; 0.0625 g.kg(-1) LTM.d(-1) x 49 d).nnnRESULTSnBiopsy samples indicated that total creatine (TCr=free Cr + PCr) was significantly lower in VG compared with NV at baseline (VG=117 mmol.kg(-1); NV=130 mmol.kg(-1); P<0.05). For Cr subjects, there was a greater increase in PCr, TCr, bench-press strength, isokinetic work, Type II fiber area, and whole-body lean tissue compared with subjects on placebo (P<0.05). Vegetarians who took Cr had a greater increase in TCr, PCr, lean tissue, and total work performance than nonvegetarians who took Cr (P<0.05). The change in muscle TCr was significantly correlated with initial muscle TCr, and the change in lean tissue mass and exercise performance. These findings confirm an ergogenic effect of Cr during resistance training and suggest that subjects with initially low levels of intramuscular Cr (vegetarians) are more responsive to supplementation.

Effects of high‐intensity endurance exercise training in the G93A mouse model of amyotrophic lateral sclerosis

The G93A transgenic mouse has a mutation in copper/zinc superoxide dismutase (CuZnSOD) that results in oxidative stress and motor neuron loss. Endurance exercise training is known to increase antioxidant capacity in skeletal muscle. Therefore, we hypothesized that endurance training may extend onset of disease or survival in the G93A mouse. We examined the effects of high‐intensity endurance exercise training (45 min/day, 5 times/week, progressive increase from 9 to 22 m/min) on disease onset and survival in G93A mice. Endurance training did not affect clinical onset, although it hastened death in male mice (P < 0.05). Endurance‐trained males had a statistically significant decrease in rotarod performance at 112 days (P < 0.05), whereas sedentary males decreased at 119 days (P < 0.05). Endurance‐trained and sedentary females decreased at 126 days and 129 days, respectively (P < 0.05). Female mice lived longer than males (P < 0.05), and there was a trend for hastened clinical onset in males (P = 0.062). We conclude that high‐intensity endurance exercise training does not affect onset of clinical symptoms in G93A mice but hastens a decrease in motor performance and death following onset of clinical symptoms in male mice only. In light of a recent report describing increased survival following low‐intensity endurance training, it appears that training intensity is an important determinant of survival in the G93A mouse.

Gene expression profiling in human skeletal muscle during recovery from eccentric exercise

We used cDNA microarrays to screen for differentially expressed genes during recovery from exercise-induced muscle damage in humans. Male subjects (n = 4) performed 300 maximal eccentric contractions, and skeletal muscle biopsy samples were analyzed at 3 h and 48 h after exercise. In total, 113 genes increased 3 h postexercise, and 34 decreased. At 48 h postexercise, 59 genes increased and 29 decreased. On the basis of these data, we chose 19 gene changes and conducted secondary analyses using real-time RT-PCR from muscle biopsy samples taken from 11 additional subjects who performed an identical bout of exercise. Real-time RT-PCR analyses confirmed that exercise-induced muscle damage led to a rapid (3 h) increase in sterol response element binding protein 2 (SREBP-2), followed by a delayed (48 h) increase in the SREBP-2 gene targets Acyl CoA:cholesterol acyltransferase (ACAT)-2 and insulin-induced gene 1 (insig-1). The expression of the IL-1 receptor, a known regulator of SREBP-2, was also elevated after exercise. Taken together, these expression changes suggest a transcriptional program for increasing cholesterol and lipid synthesis and/or modification. Additionally, damaging exercise induced the expression of protein kinase H11, capping protein Z alpha (capZalpha), and modulatory calcineurin-interacting protein 1 (MCIP1), as well as cardiac ankryin repeat protein 1 (CARP1), DNAJB2, c-myc, and junD, each of which are likely involved in skeletal muscle growth, remodeling, and stress management. In summary, using DNA microarrays and RT-PCR, we have identified novel genes that respond to skeletal muscle damage, which, given the known biological functions, are likely involved in recovery from and/or adaptation to damaging exercise.

Treadmill training–induced adaptations in muscle phenotype in persons with incomplete spinal cord injury

Body weight–supported treadmill (BWST) training has been shown to improve ambulatory capacity in persons with a spinal cord injury (SCI); however, the effect that BWST training has on skeletal muscle phenotype is unknown. We aimed to determine whether 6 months (three sessions/week) of BWST training in neurologically stable persons with a traumatic spinal cord injury (ASIA C) alters skeletal muscle phenotype, ambulatory capacity, and blood lipid profile. Externally supported body weight decreased, and walking velocity and duration of the training sessions increased (all P < 0.05) as a result of training. Muscle biopsies revealed increases in the mean muscle‐fiber area of type I and IIa fibers. Training induced a reduction in type IIax/IIx fibers, as well as a decrease in IIX myosin heavy chain, and an increase in type IIa fibers. Maximal citrate synthase and 3‐hydroxy‐acyl‐CoA dehydrogenase activity also increased following training. BWST training brought about reductions in plasma total (−11%) and low‐density lipoprotein (−13%) cholesterol. We conclude that, in patients with a spinal cord injury, BWST training is able to induce an increase in muscle fiber size and bring about increases in muscle oxidative capacity. In addition, BWST training can bring about improvements in ambulatory capacity and antiatherogenic changes in blood lipid profile. Muscle Nerve 30: 61–68, 2004

Oxidative stress and antioxidant enzyme upregulation in SOD1‐G93A mouse skeletal muscle

Amyotrophic lateral sclerosis (ALS) is caused by motor neuron loss in the spinal cord, but the mechanisms responsible are not known. Ubiquitous transgenic overexpression of copper/zinc superoxide dismutase (SOD1) mutations causing familial ALS (SOD1mut) leads to an ALS phenotype in mice; however, restricted expression of SOD1mut in neurons alone is not sufficient to cause this phenotype, suggesting that non‐neuronal SOD1mut expression is also required for disease manifestation. Recently, several investigators have suggested that SOD1mut‐mediated oxidative stress in skeletal muscle may contribute to ALS pathogenesis. The purpose of this study was to examine oxidative stress and antioxidant enzyme adaptation in 95‐day‐old SOD1‐G93A skeletal muscle. We observed significant elevations in both malondialdehyde (22% and 31% in red and white gastrocnemius, respectively) and protein carbonyls (53% in red gastrocnemius) in SOD1‐G93A mice. Copper/zinc SOD activity was higher in red and white SOD1‐G93A gastrocnemius (7‐ and 10‐fold, respectively), as was manganese SOD (4‐ and 5‐fold, respectively) and catalase (2‐ and 2.5‐fold, respectively). Taken together, our data demonstrate oxidative stress and compensatory antioxidant enzyme upregulation in SOD1‐G93A skeletal muscle. Muscle Nerve, 2006

Creatine monohydrate supplementation does not increase muscle strength, lean body mass, or muscle phosphocreatine in patients with myotonic dystrophy type 1.

Creatine monohydrate (CrM) supplementation may increase strength in some types of muscular dystrophy. A recent study in myotonic muscular dystrophy type 1 (DM1) did not find a significant treatment effect, but measurements of muscle phosphocreatine (PCr) were not performed. We completed a randomized, double‐blind, cross‐over trial using 34 genetically confirmed adult DM1 patients without significant cognitive impairment. Participants received CrM (5 g, ∼0.074 g/kg daily) and a placebo for each 4‐month phase with a 6‐week wash‐out. Spirometry, manual muscle testing, quantitative isometric strength testing of handgrip, foot dorsiflexion, and knee extension, handgrip and foot dorsiflexion endurance, functional tasks, activity of daily living scales, body composition (total, bone, and fat‐free mass), serum creatine kinase activity, serum creatinine concentration and clearance, and liver function tests were completed before and after each intervention, and muscle PCr/β‐adenosine triphosphate (ATP) ratios of the forearm flexor muscles were completed at the end of each phase. CrM supplementation did not increase any of the outcome measurements except for plasma creatinine concentration (but not creatinine clearance). Thus, CrM supplementation at 5 g daily does not have any effects on muscle strength, body composition, or activities of daily living in patients with DM1, perhaps because of a failure of the supplementation to increase muscle PCr/β‐ATP content. Muscle Nerve 29: 51–58, 2004

Novel SCO2 mutation (G1521A) presenting as a spinal muscular atrophy type I phenotype

Rare cases of suspected spinal muscular atrophy (SMA) have been found to have cytochrome c oxidase (COX) deficiency. To date, four cases with SMA features have been reported in children with mutations in the synthesis of cytochrome oxidase 2 (SCO2) gene. We report a male neonate who was born hypotonic, with persistent lactic acidosis, spontaneous activity with EMG testing, development of respiratory distress in the first few hours of life, and died at 30 days of age with progressive cardiomyopathy. Testing for survival motor neurone (smn) and NAIP deletions were negative and a skeletal muscle biopsy showed neurogenic features with severe reductions of COX enzymatic and histochemical staining intensity. Post‐mortem muscle, heart, and liver biopsies showed severe, moderate, and mild reductions in COX activity, respectively, with parallel findings in the protein content for the mitochondrial DNA (COII) and nuclear DNA (COIV) encoded subunits. DNA sequencing of exon 2 of the SCO2 gene revealed compound heterozygosity with mutations at G1541A (common mutation, E140K) and also at a novel site in the copper binding region (G1521A in the current case (converting a highly conserved cysteine to serine (C133S)); mother heterozygous for G1521A; and father heterozygous for G1541A). This case provides strong support that SCO2 mutations can result in neonatal hypotonia with an SMA 1 phenotype. SCO2 mutations should be screened in suspected SMA cases with normal smn mutation analysis and any one of; cardiomyopathy, lactic acidosis, or COX deficiency in muscle.

Diagnostic utility of a modified forearm ischemic exercise test and technical issues relevant to exercise testing.

The sensitivity and specificity of a modified forearm ischemic test (FIT) are described in the diagnosis of glycogen storage disease, myoadenylate deaminase deficiency, and mitochondrial disease. FIT and muscle biopsy results were reviewed from 99 patients (glycogen storage disease [GSD], myoadenylate deaminase deficiency [AMPD], mitochondrial disease [MITO], miscellaneous neuromuscular disorders, and controls). The influence of catheter placement and an antecedent sugar bolus were also assessed in healthy young men. The FIT had a sensitivity of 1.00 and a specificity of 1.00 for a diagnosis of GSD, whereas the corresponding values were 1.00 and 0.37 for AMPD deficiency. A baseline lactate of >2.5 mmol/L provided the highest sensitivity (0.62) and specificity (1.00) for MITO disease. A baseline and +1 min sample provided optimal sensitivity and specificity for GSD and AMPD deficiency. Catheter placement in any vein other than the ipsilateral antecubital resulted in attenuated lactate responses (P < 0.0001). A pre‐FIT sugar bolus did not alter the postexercise lactate or ammonia response. Thus, a modified FIT was helpful in the diagnosis of GSD and excluding AMPD deficiency, but not in the diagnosis of MITO disease. Catheter placement is critical to the interpretation of a FIT, whereas pretesting diet is less important. Muscle Nerve 27: 359–366, 2003