Cardiac and skeletal muscle manifestations in the G608G mouse model of Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disorder resulting from de novo mutations in the lamin A gene. Children with HGPS typically pass away in their teenage years due to cardiovascular diseases such as atherosclerosis, myocardial infarction, heart failure, and stroke. In this study, we characterized the G608G HGPS mouse model and explored cardiac and skeletal muscle function, along with senescence-associated phenotypes in fibroblasts. Homozygous G608G HGPS mice exhibited cardiac dysfunction, including decreased cardiac output and stroke volume, and impaired left ventricle relaxation. Additionally, skeletal muscle exhibited decreased isometric tetanic torque, muscle atrophy, and increased fibrosis. HGPS fibroblasts showed nuclear abnormalities, decreased proliferation, and increased expression of senescence markers. These findings provide insights into the pathophysiology of the G608G HGPS mouse model and inform potential therapeutic strategies for HGPS.

Diet modification reverses diastolic dysfunction in rats with heart failure and preserved ejection fraction.

Dahl Salt-Sensitive (DSS) rats develop heart failure with preserved ejection fraction (HFpEF) when fed a high-salt (8 % NaCl) diet. Hypertension-induced inflammation and subsequent ventricular fibrosis are believed to underlie the development of HFpEF. We investigated the role of diet modification in the progression of HFpEF using male DSS rats, fed either a high-salt diet from7 weeks of age to induce HFpEF, ora normal-salt (0.3% NaCl) diet as controls. After echocardiographic confirmation of diastolic dysfunction at 14-15 weeks of age along with HF manifestations, the HFpEF rats were randomly assigned to either continue a high-salt diet or switch to a normal-salt diet for an additional 4 weeks. HFpEF rats with diet modification showed improved diastolic function (reduced E/E' ratio in echocardiogram), increased functional capacity (increased treadmill exercise distance), and reduced pulmonary congestions (lung/body weight ratio), compared to high-salt-fed HFpEF rats. Systolic blood pressure remained high (~200 mmHg), and ventricular hypertrophy remained unchanged. Ventricular arrhythmia inducibility (100 % inducible) and corrected QT interval (on ECG) did not change in HFpEF rats after diet modification. HFpEF rats with diet modification showed prolonged survival and reduced ventricular fibrosis (Masson's trichrome staining) compared to high-salt-fed HFpEF rats. Hence, the modification of diet (from high-salt to normal-salt diet) reversed HFpEF phenotypes without affecting blood pressure or ventricular hypertrophy.

Casein-enhanced uptake and disease-modifying bioactivity of ingested extracellular vesicles.

Extracellular vesicles (EVs) from cardiac stromal cells, developed as therapeutic candidates, improve dystrophic muscle function when administered parenterally, but oral delivery remains untested. We find that casein, the dominant protein in breast milk, enhances the uptake and bioactivity of ingested heart-derived EVs, altering gene expression in blood cells and enhancing muscle function in mdx mice with muscular dystrophy. Thus, EVs, administered orally, are absorbed and exert disease-modifying bioactivity in vivo. Formulating EVs with casein enhances uptake and markedly expands the range of potential therapeutic applications.

Cardiosphere-derived cells, with and without a biological scaffold, stimulate myogenesis and recovery of muscle function in mice with volumetric muscle loss.

Extremity trauma to military personnel and civilians commonly results in volumetric muscle loss (VML), leaving patients suffering chronic physical disability. Biomaterial-based technologies such as extracellular matrices (ECMs) are currently in clinical testing for soft tissue repair, but, in preclinical models of VML, the efficacy of ECMs is equivocal. In a murine model of VML, we investigated the effects of ECM and/or cardiosphere-derived cell (CDC) therapy; the latter improves skeletal myogenesis and muscle function in mdx mice, so we reasoned that CDCs may exert disease-modifying bioactivity in VML. While ECM alone improves functional recovery, CDCs have no additive or synergistic benefits with ECM transplantation following VML injury. However, CDCs alone are sufficient to promote muscle recovery, leading to sustained increases in muscle function throughout the study period. Notably, CDCs stimulate satellite cell accumulation in the muscle defect area and hasten myogenic progression (as evidenced by qPCR gene expression profiling), leading to global increases in myofiber numbers and anterior muscle compartment volume. Together, these data implicate CDCs as a viable therapeutic candidate to regenerate skeletal muscle injured by VML.

Disease-modifying bioactivity of intravenous cardiosphere-derived cells and exosomes in mdx mice.

Dystrophin deficiency leads to progressive muscle degeneration in Duchenne muscular dystrophy (DMD) patients. No known cure exists, and standard care relies on the use of antiinflammatory steroids, which are associated with side effects that complicate long-term use. Here, we report that a single intravenous dose of clinical-stage cardiac stromal cells, called cardiosphere-derived cells (CDCs), improves the dystrophic phenotype in mdx mice. CDCs augment cardiac and skeletal muscle function, partially reverse established heart damage, and boost the regenerative capacity of skeletal muscle. We further demonstrate that CDCs work by secreting exosomes, which normalize gene expression at the transcriptome level, and alter cell signaling and biological processes in mdx hearts and skeletal muscle. The work reported here motivated the ongoing HOPE-2 clinical trial of systemic CDC delivery to DMD patients, and identifies exosomes as next-generation cell-free therapeutic candidates for DMD.

Augmenting canonical Wnt signalling in therapeutically inert cells converts them into therapeutically potent exosome factories.

Cardiosphere-derived cells are therapeutic candidates with disease-modifying bioactivity, but their variable potency has complicated their clinical translation. Transcriptomic analyses of cardiosphere-derived cells from human donors have revealed that their therapeutic potency correlates with Wnt/ß-catenin signalling and with ß-catenin protein levels. Here, we show that skin fibroblasts engineered to overexpress ß-catenin and the transcription factor Gata4 become immortal and therapeutically potent. Transplantation of the engineered fibroblasts into a mouse model of acute myocardial infarction led to improved cardiac function and mouse survival, and in the mdx mouse model of Duchenne muscular dystrophy, exosomes secreted by the engineered fibroblasts improved exercise capacity and reduced skeletal-muscle fibrosis. We also demonstrate that exosomes from high-potency cardiosphere-derived cells exhibit enhanced levels of miR-92a (a known potentiator of the Wnt/ß-catenin pathway), and that they activate cardioprotective bone-morphogenetic-protein signalling in cardiomyocytes. Our findings show that the modulation of canonical Wnt signalling can turn therapeutically inert mammalian cells into immortal exosome factories for cell-free therapies.

Exosome-Mediated Benefits of Cell Therapy in Mouse and Human Models of Duchenne Muscular Dystrophy.

Genetic deficiency of dystrophin leads to disability and premature death in Duchenne muscular dystrophy (DMD), affecting the heart as well as skeletal muscle. Here, we report that clinical-stage cardiac progenitor cells, known as cardiosphere-derived cells (CDCs), improve cardiac and skeletal myopathy in the mdx mouse model of DMD. Injection of CDCs into the hearts of mdx mice augments cardiac function, ambulatory capacity, and survival. Exosomes secreted by human CDCs reproduce the benefits of CDCs in mdx mice and in human induced pluripotent stem cell-derived Duchenne cardiomyocytes. Surprisingly, CDCs and their exosomes also transiently restored partial expression of full-length dystrophin in mdx mice. The findings further motivate the testing of CDCs in Duchenne patients, while identifying exosomes as next-generation therapeutic candidates.

Skeletal muscle adaptations to testosterone and resistance training in men with COPD.

We recently reported increased leg lean mass and strength in men with chronic obstructive pulmonary disease (COPD) receiving 10 wk of testosterone (T) and leg resistance training (R) (Casaburi R, Bhasin S, Cosentino L, Porszasz J, Somfay A, Lewis M, Fournier M, Storer T. Am J Respir Crit Care Med 170: 870-878, 2004). The present study evaluates the role of muscle IGF and related factors as potential mechanisms for our findings, using quadriceps muscle biopsies from the same cohort. Patient groups were 1) weekly placebo (P) injections + no R; 2) P and R; 3) weekly injections of T + no R; and 4) T + R (TR). Muscle fibers were classified histochemically, and their cross-sectional areas (CSAs) and fiber density (number of fibers per unit area) were determined. Gene transcripts were determined by real-time PCR and protein expression by RIA. While no significant changes in fiber CSAs were noted across groups, increased trends were observed after 10 wk, and significant decrements in muscle fiber density were noted in all treated groups. A global increase in all myosin heavy chain (MyHC) mRNA isoforms was observed in TR patients. Muscle IGF-IEa and IGF-IEc mRNAs were significantly increased with TR group. Muscle IGF-I protein was increased in all intervention groups (greatest in TR). While TR IGF-II mRNA was increased, protein levels were unaltered. IGF binding protein-4 mRNA was increased with TR. Myogenin mRNA was increased in both T groups, while MyoD and myostatin were unchanged. Muscle atrophy F-box mRNA tended to increase with TR. Our data suggest that the combined interventions produced an enhanced local anabolic milieu driven in large part by the muscle IGF system, despite potentially negative biochemical influences present in COPD patients.

Therapeutic benefits of intravenous cardiosphere-derived cell therapy in rats with pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a progressive condition characterized by occlusive pulmonary arteriopathy, in which survival remains poor despite pharmacologic advances. The aim of this study was to evaluate the ability of cardiosphere-derived cells (CDCs), cardiac progenitor cells with potent anti-inflammatory and immunomodulatory properties, to attenuate hemodynamic and morphometric remodeling of the right ventricle (RV) and pulmonary arterioles in rats with established monocrotaline (MCT)-induced PAH. Animals were divided into 3 groups: 1) Control (CTL), 2) PAH in which CDCs were centrally infused (CDC) and 3) PAH in which saline was given (Sham). Significant increments in RV systolic pressure (RVSP) and RV hypertrophy were noted in Sham animals compared to CTL. In CDC rats at day 35, RSVP fell (- 38%; p< 0.001) and RV hypertrophy decreased (-26%; p< 0.01). TAPSE and cardiac output were preserved in all 3 groups at day 35. Pulmonary arteriolar wall thickness was greater in Sham rats compared to CTL, and reduced in CDC animals for vessels 20-50 µm (P<0.01; back to CTL levels) and 50-80µm (P<0.01) in diameter. The macrophage population was increased in Sham animals compared to CTL (P< 0.001), but markedly reduced in CDC rats. In conclusion, infusion of CDCs markedly attenuated several key pathophysiologic features of PAH. As adjunctive therapy to PAH-specific agents, CDCs have the potential to impact on the pathobiology of adverse pulmonary arteriolar remodeling, by acting on multiple mechanisms simultaneously.

Effect of severe short-term malnutrition on diaphragm muscle signal transduction pathways influencing protein turnover.

The aim of this study was to evaluate the effect of nutritional deprivation (ND) on signal transduction pathways influencing the translational apparatus in the diaphragm muscle. Male rats were divided into two groups: 1) 20% of usual food intake for 4 days (ND) with water provided at libitum and 2) free-eating control (Ctl). Total protein and RNA were extracted from the diaphragm. Insulin-like growth factor I mRNA was analyzed by RT-PCR. Protein analyses of key cytoplasmic proteins for three signaling pathways deemed important in influencing protein turnover [phosphatidylinositol 3-kinase- Akt-mammalian target of rapamycin, P13K/Akt/glycogen synthase kinase (GSK)-3, and MAPK-ERK] were performed by Western blot. Body weight decreased 30% in ND and increased 17% in Ctl animals. Diaphragm mass decreased 29% in ND animals. Muscle insulin-like growth factor I mRNA abundance was reduced 63% in ND animals. ND resulted in a 55% reduction in phosphorylated (Ser473) Akt. Phosphorylation of mammalian target of rapamycin at Ser2448 was reduced by 85% in ND animals. Downstream effectors important in translation initiation were also affected by ND. Phosphorylated (Thr389) 70-kDa ribosomal protein S6 kinase was significantly reduced (35%) by ND. ND also resulted in significant dephosphorylation of the translational repressor initiation factor 4E-binding protein 1. Phosphorylation of GSK-3alpha (Ser21) and GSK-3beta (Ser9) was increased 55 and 45%, respectively, with ND. Phosphorylation of ERK1 (Thr202) and ERK2 (Tyr204), p44 and p42, respectively, was reduced 64 and 55%, respectively, with ND. Total protein concentration for all signaling intermediates of the three pathways was preserved. We conclude that short-term ND altered the phosphorylation states of key proteins of several pathways involved in protein turnover. This forms the framework for future studies aimed at identifying therapeutic targets in the management of short-term nutritionally induced cachectic states.

Transcriptional levels of growth factors in skeletal muscle of maintenance hemodialysis patients.

This study was performed to assess the mRNA levels in skeletal muscle of maintenance hemodialysis (MHD) patients. Fifty-one sedentary MHD patients and 21 sedentary normal adults of similar age, gender distribution, and racial/ethnic mix as the patients were examined. The subjects had needle muscle biopsy to measure mRNA levels. They were assessed in right vastus lateralis muscle for insulinlike growth factor I (IGF-IEa and IGF-IEc), IGF-II, the IGF-I receptor, the IGF-II receptor, and myostatin. mRNA was measured by real-time polymerase chain reaction amplification of reverse transcribed cDNA. The results showed that in the MHD patients, as compared with the normal control patients, skeletal muscle mRNA levels for IGF-IEa, IGF-II, and the IGF-I receptor were significantly reduced, whereas mRNA for IGF-IEc, IGF-II receptor, and myostatin were not different than normal. We conclude that sedentary MHD patients show reductions in mRNA levels in the right vastus lateralis muscle for certain growth factor proteins, notably IGF-IEa, IGF-II, and the IGF-I receptor. These abnormalities may contribute to the sarcopenia and impaired endurance capacity, strength, and physical performance that occur in MHD patients.

Effect of exercise on mRNA levels for growth factors in skeletal muscle of hemodialysis patients.

OBJECTIVES: Muscle mass and muscle mRNA levels for certain growth factors are reduced in maintenance hemodialysis (MHD) patients. This study tested the hypothesis that in MHD patients endurance exercise training (EET) increases mRNA levels for insulin-like growth factors and reduces myostatin mRNA. DESIGN: Biopsies of the right vastus lateralis muscle were performed before and at the end of 8.9 +/- 0.9 (SEM) weeks of EET in MHD patients. Muscle tissue was analyzed histologically by electron microscopy and for fiber cross-sectional area, and, in 8 pairs of biopsies, muscle was examined for mRNA levels for the following proteins: myostatin, insulin-like growth factor-I (IGF-I), IGF-I receptor (IGF-IR), IGF binding proteins (IGFBPs)-1, -2, -3, -4, and -5, and IGF-binding protein-related protein-1 (IGFBP-rP1). SETTING: Outpatient MHD centers. PATIENTS: This was a pilot study conducted in sedentary clinically stable MHD patients undergoing EET with no control group. INTERVENTION: EET that was carefully supervised by exercise trainers. MAIN OUTCOME MEASURE: Skeletal muscle mRNA levels, especially myostatin mRNA. RESULTS: With EET, skeletal muscle myostatin mRNA decreased by 51%, mRNA levels increased significantly for IGF-IR (by 41%), IGFBP-2, -4, and -5, and IGFBP-rP1. IGF-I mRNA increased by 35%; this change was not significant. IGFBP-3 mRNA did not change, and IGFBP-1 mRNA was undetectable. There were mild to moderate alterations in skeletal muscle ultrastructure that did not change significantly with EET. Muscle fiber size, measured in 5 patients, did not change. CONCLUSION: In MHD patients who undergo approximately 9 weeks of EET, skeletal muscle mRNA for myostatin decreases and mRNA for IGF-IR, IGFBPs -2, -4, and -5 and IGFBP-rP1 increases. These changes may indicate mechanisms by which EET improves muscle exercise capacity in MHD patients.

Tumor necrosis factor-alpha and malnutrition-induced inhibition of diaphragm fiber growth in young rats.

Tumor necrosis factor (TNF)-alpha has been implicated in several muscle-wasting disorders, with increased levels of the cytokine reported in malnourished children. The role of TNF-alpha in mediating malnutrition-induced inhibition of diaphragm (DIA) muscle growth in young growing rats was evaluated. Three groups of rats were studied: 1) control (CTL); 2) nutritional deprivation (ND; 50% of normal food intake for 7 days); and 3) ND + rat specific anti-TNF-alpha antibody. DIA fiber cross-sectional areas were determined. Serum and muscle TNF-alpha levels were measured by real-time PCR, ELISA, and immunohistochemistry. Body weights decreased 20% in ND rats and increased 46% in CTL animals. Anti-TNF-alpha had no effect on body weight or on DIA mass in ND animals. ND significantly reduced cross-sectional areas of all fiber types (33-46%). Anti-TNF-alpha failed to attenuate ND-induced inhibition of DIA fiber growth. Serum TNF-alpha levels increased 2.6-fold in ND animals, with levels suppressed to below CTL values with anti-TNF-alpha. DIA TNF-alpha mRNA and protein levels increased two- to threefold in ND rats. Anti-TNF-alpha antibodies suppressed muscle levels of the cytokine in ND animals to near CTL values. TNF-alpha immunoreactivity in all DIA fibers revealed similar directions of change in both ND groups. Direction and magnitude of change in DIA phosphorylated p38 MAPK (a likely second messenger of TNF-alpha) tracked those of TNF-alpha. Muscle levels of IGF-I mRNA and phosphorylated Akt were markedly reduced in ND animals with no change following anti-TNF-alpha therapy. Thus rat anti-TNF-alpha at a dose known to neutralize the cytokine failed to attenuate or reverse ND-induced inhibition of DIA fiber growth in our model.

Effect of endurance and/or strength training on muscle fiber size, oxidative capacity, and capillarity in hemodialysis patients.

We previously reported reduced limb muscle fiber succinate dehydrogenase (SDH) activity and capillarity density and increased cross-sectional areas (CSAs) of all fiber types in maintenance hemodialysis (MHD) patients compared with matched controls that may contribute to their effort intolerance and muscle weakness. This study evaluated whether endurance training (ET), strength training (ST), or their combination (EST) alters these metabolic and morphometric aberrations as a mechanism for functional improvement. Five groups were evaluated: 1) controls; 2) MHD/no training; 3) MHD/ET; 4) MHD/ST; and 5) MHD/EST. Training duration was 21.5 ± 0.7 wk. Vastus lateralis muscle biopsies were obtained after HD at baseline and at study end. Muscle fibers were classified immunohistochemically, and fiber CSAs were computed. Individual fiber SDH activity was determined by a microdensitometric assay. Capillaries were identified using antibodies against endothelial cells. Type I and IIA fiber CSAs decreased significantly (10%) with EST. In the ET group, SDH activity increased 16.3% in type IIA and 19.6% in type IIX fibers. Capillary density increased significantly by 28% in the EST group and 14.3% with ET. The number of capillaries surrounding individual fiber type increased significantly in EST and ET groups. Capillary-to-fiber ratio increased significantly by 11 and 9.6% in EST and ET groups, respectively. We conclude that increments in capillarity and possibly SDH activity in part underlie improvements in endurance of MHD patients posttraining. We speculate that improved specific force and/or neural adaptations to exercise underlie improvements in limb muscle strength of MHD patients.

Influence of varying degrees of malnutrition on IGF-I expression in the rat diaphragm.

This study evaluated the impact of varying degrees of prolonged malnutrition on the local insulin-like growth factor-I (IGF-I) system in the costal diaphragm muscle. Adult rats were provided with either 60 or 40% of usual food intake over 3 wk. Nutritionally deprived (ND) animals (i.e., ND60 and ND40) were compared with control (Ctl) rats fed ad libitum. Costal diaphragm fiber types and cross-sectional areas were determined histochemically. Costal diaphragm muscle IGF-I mRNA levels were determined by RT-PCR. Serum and muscle IGF-I peptide levels were determined by using a rat-specific radioimmunoassay. The body weights of Ctl rats increased by 5%, whereas those of ND60 and ND40 animals decreased by 16 and 26%, respectively. Diaphragm weights were reduced by 17 and 27% in ND60 and ND40 animals, respectively, compared with Ctl. Diaphragm fiber proportions were unaffected by either ND regimen. Significant atrophy of both type IIa and IIx fibers was noted in the ND60 group, whereas atrophy of all three fiber types was observed in the diaphragm of ND40 rats. Serum IGF-I levels were reduced by 62 and 79% in ND60 and ND40 rats, respectively, compared with Ctl. Diaphragm muscle IGF-I mRNA levels in both ND groups were similar to those noted in Ctl. In contrast, IGF-I concentrations were reduced by 36 and 42% in the diaphragm muscle of ND60 and ND40 groups, respectively, compared with Ctl. We conclude that the local (autocrine/paracrine) muscle IGF-I system is affected in our models of prolonged ND. We propose that this contributes to disordered muscle protein turnover and muscle cachexia with atrophy of muscle fibers. This is particularly so in view of recent data demonstrating the importance of the autocrine/paracrine system in muscle growth and maintenance of fiber size.

Arterial injury in mice with severe insulin-like growth factor-1 (IGF-1) deficiency.

BACKGROUND: Insulin-like growth factor-1 plays a significant role in wound healing. Injury to the arterial wall is followed by a marked increase in insulin-like growth factor-1 expression and inhibition of insulin-like growth factor-1 action is associated with diminished intimal thickening after injury. METHODS AND RESULTS: The role of insulin-like growth factor-1 in arterial response to cuff injury was investigated in genetically modified mice with severe insulin-like growth factor-1 deficiency ((m/m) mice). Tissue and serum insulin-like growth factor-1 was severely decreased, by 40% to 60% before the injury and by 50% to 60% following the arterial injury in insulin-like growth factor-1 (m/m) mice compared to control mice. Nevertheless, following the cuff induced injury to the carotid arteries, insulin-like growth factor-1 (m/m) mice had a similar number of proliferating medial cells 3 days after injury and similar neointimal thickening (0.019 +/- 0.015 C57BL/6J vs. 0.016 +/- 0.014 mm(2), P = 0.26) 21 days after injury compared to wild type C57BL/6J mice. The phases of the response to injury that are mediated by insulin-like growth factor-1 were studied with recombinant human insulin-like growth factor-1 in rats with balloon-injured femoral arteries. Treatment of rats with recombinant human insulin-like growth factor-1 increased neointimal thickening (0.0265 +/- 0.0099 vs 0.0156 +/- 0.0049 mm(2), P = 0.03), intimal smooth muscle cell numbers (195.6 +/- 40.2 vs 145.3 +/- 27.3; P = 0.03), and the ratio of proliferating intimal to medial smooth muscle cells (10.7 +/- 6.9 vs 3.0 +/- 2.1; P = 0.03) 7 days after injury compared to untreated rats. At 14 days neointimal area was similar in the 2 groups of rats. CONCLUSIONS: The data in insulin-like growth factor-1 deficient mice suggest a relatively low threshold tissue concentration for insulin-like growth factor-1 to exhibit its role in vascular response to injury. The findings in rats treated with recombinant human insulin-like growth factor-1 suggest that insulin-like growth factor-1 is primarily involved in the early phases of neointimal formation.

Effects of malnutrition with or without eicosapentaenoic acid on proteolytic pathways in diaphragm.

Attenuation of muscle wasting has been reported with eicosapentaenoic acid (EPA) use in cachectic states. Pathways mediating muscle proteolysis with severe short-term nutritional deprivation (ND)±EPA were evaluated, including diaphragm fiber-specific cross-sectional areas, mRNA (real-time PCR) and protein expression (Western blot). Rats were divided into three groups: (1) free-eating controls, (2) ND and (3) ND+EPA. ND significantly influenced multiple proteolytic pathways. EPA significantly reduced mRNA abundances for most genes to control levels with ND. However, discordant muscle protein expression of many genes was noted with the use of EPA, as protein levels failed to fall. EPA had no impact on diaphragm muscle atrophy, despite the impressive mRNA and some protein results. We conclude that EPA does not attenuate diaphragm muscle atrophy with severe levels of ND. Postulated mechanisms include reduction in muscle protein synthesis and persistent ongoing stimuli for proteolysis. Our study provides unique data on proteolytic signals with ND and has important implications for future studies using EPA.

Metabolic and morphometric profile of muscle fibers in chronic hemodialysis patients.

Muscle weakness and effort intolerance are common in maintenance hemodialysis (MHD) patients. This study characterized morphometric, histochemical, and biochemical properties of limb muscle in MHD patients compared with controls (CTL) with similar age, gender, and ethnicity. Vastus lateralis muscle biopsies were obtained from 60 MHD patients, 1 day after dialysis, and from 21 CTL. Muscle fiber types and capillaries were identified immunohistochemically. Individual muscle fiber cross-sectional areas (CSA) were quantified. Individual fiber oxidative capacities were determined (microdensitometric assay) to measure succinate dehydrogenase (SDH) activity. Mean CSAs of type I, IIA, and IIX fibers were 33, 26, and 28% larger in MHD patients compared with CTL. SDH activities for type I, IIA, and IIX fibers were reduced by 29, 40, and 47%, respectively, in MHD. Capillary to fiber ratio was increased by 11% in MHD. The number of capillaries surrounding individual fiber types were also increased (type I: 9%; IIA: 10%; IIX: 23%) in MHD patients. However, capillary density (capillaries per unit muscle fiber area) was reduced by 34% in MHD patients, compared with CTL. Ultrastuctural analysis revealed swollen mitochondria with dense matrix in MHD patients. These results highlight impaired oxidative capacity and capillarity in MHD patients. This would be expected to impair energy production as well as substrate and oxygen delivery and exchange and contribute to exercise intolerance. The enlarged CSA of muscle fibers may, in part, be accounted for by edema. We speculate that these changes contribute to reduce limb strength in MHD patients by reducing specific force.