Roberto Bottinelli

Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs.

Duchenne muscular dystrophy remains an untreatable genetic disease that severely limits motility and life expectancy in affected children. The only animal model specifically reproducing the alterations in the dystrophin gene and the full spectrum of human pathology is the golden retriever dog model. Affected animals present a single mutation in intron 6, resulting in complete absence of the dystrophin protein, and early and severe muscle degeneration with nearly complete loss of motility and walking ability. Death usually occurs at about 1 year of age as a result of failure of respiratory muscles. Here we report that intra-arterial delivery of wild-type canine mesoangioblasts (vessel-associated stem cells) results in an extensive recovery of dystrophin expression, normal muscle morphology and function (confirmed by measurement of contraction force on single fibres). The outcome is a remarkable clinical amelioration and preservation of active motility. These data qualify mesoangioblasts as candidates for future stem cell therapy for Duchenne patients.

FORCE-VELOCITY RELATIONS AND MYOSIN HEAVY CHAIN ISOFORM COMPOSITIONS OF SKINNED FIBRES FROM RAT SKELETAL MUSCLE

1. This study was performed to assess whether muscle contractile properties are related to the presence of specific myosin heavy chain (MHC) isoforms. 2. Force‐velocity relations and MHC isoform composition were determined in seventy‐four single skinned muscle fibres from rat soleus, extensor digitorum longus and plantaris muscles. 3. Four groups of fibres were identified according to their MHC isoform composition determined by monoclonal antibodies: type 1 (slow), and types 2A, 2B and 2X (fast). 4. With respect to maximum velocity of shortening (V0), the fibres formed a continuum between 0.35 and 2.84 L/s (muscle lengths per second) at 12 degrees C. V0 in type 1 fibres (slow fibres) was between 0.35 and 0.95 L/s (0.639 +/‐ 0.038 L/s; mean +/‐ S.E. of mean). V0 in type 2 fibres (fast fibres) was consistently higher than 0.91 L/s. Ranges of V0 in the three fast fibre types mostly overlapped. Type 2A and 2X fibres had similar mean V0 values (1.396 +/‐ 0.084 and 1.451 +/‐ 0.066 L/s respectively); type 2B fibres showed a higher mean V0 value (1.800 +/‐ 0.109 L/s) than type 2A and 2X fibres. 5. Mean values of a/P0, an index of the curvature of force‐velocity relations, allowed us to identify two groups of fibres: a high curvature group comprised of type 1 (mean a/P0, 0.066 +/‐ 0.007) and 2A (0.066 +/‐ 0.024) fibres and a low curvature group comprised of type 2B (0.113 +/‐ 0.013) and 2X (0.132 +/‐ 0.008) fibres. 6. Maximal power output was lower in slow fibres than in fast fibres, and among fast fibres it was lower in type 2A fibres than in type 2X and 2B. 7. Force per unit cross‐sectional area was less in slow fibres than in fast fibres. There was no relation between fibre type and cross‐sectional area. 8. The results suggest that MHC composition is just one of the determinants of shortening velocity and of other muscle contractile properties.

Force‐velocity properties of human skeletal muscle fibres: myosin heavy chain isoform and temperature dependence.

1. A large population (n = 151) of human skinned skeletal muscle fibres has been studied. Force‐velocity curves of sixty‐seven fibres were obtained by load‐clamp manoeuvres at 12 degrees C. In each fibre maximum shortening velocity (Vmax), maximum power output (Wmax), optimal velocity (velocity at which Wmax is developed, Vopt), optimal force (force at which Wmax is developed, Popt), specific tension (Po/CSA, isometric tension/cross‐sectional area) were assessed. Unloaded shortening velocity (Vo) was also determined at 12 degrees C in a different group (n = 57) of fibres by slack‐test procedure. 2. All fibres used for mechanical experiments were characterized on the basis of the myosin heavy chain (MHC) isoform composition by sodium dodecyl sulphate (SDS)‐polyacrylamide gel electrophoresis and divided into five types: type I (or slow), types IIA and IIB (or fast), and types I‐IIA and IIA‐IIB (or mixed types). 3. Vmax, Wmax, Vopt, Popt, Vopt/Vmax ratio, Po/CSA and Vo were found to depend on MHC isoform composition. All parameters were significantly lower in type I than in the fast (type IIA and IIB) fibres. Among fast fibres, Vmax, Wmax, Vopt and Vo were significantly lower in type IIA and than in IIB fibres, whereas Popt, Po/CSA and Vopt/Vmax were similar. 4. The temperature dependence of Vo and Po/CSA was assessed in a group of twenty‐one fibres in the range 12‐22 degrees C. In a set of six fibres temperature dependence of Vmax was also studied. The Q10 (5.88) and activation energy E (125 kJ mol‐1) values for maximum shortening velocity calculated from Arrhenius plots pointed to a very high temperature sensitivity. Po/CSA was very temperature dependent in the 12‐17 degrees C range, but less dependent between 17 and 22 degrees C.

Human skeletal muscle fibres: molecular and functional diversity

Contractile and energetic properties of human skeletal muscle have been studied for many years in vivo in the body. It has been, however, difficult to identify the specific role of muscle fibres in modulating muscle performance. Recently it has become possible to dissect short segments of single human muscle fibres from biopsy samples and make them work in nearly physiologic conditions in vitro. At the same time, the development of molecular biology has provided a wealth of information on muscle proteins and their genes and new techniques have allowed analysis of the protein isoform composition of the same fibre segments used for functional studies. In this way the histological identification of three main human muscle fibre types (I, IIA and IIX, previously called IIB) has been followed by a precise description of molecular composition and functional and biochemical properties. It has become apparent that the expression of different protein isoforms and therefore the existence of distinct muscle fibre phenotypes is one of the main determinants of the muscle performance in vivo. The present review will first describe the mechanisms through which molecular diversity is generated and how fibre types can be identified on the basis of structural and functional characteristics. Then the molecular and functional diversity will be examined with regard to (1) the myofibrillar apparatus; (2) the sarcolemma and the sarcoplasmic reticulum; and (3) the metabolic systems devoted to producing ATP. The last section of the review will discuss the advantage that fibre diversity can offer in optimizing muscle contractile performance.

The effect of ageing and immobilization on structure and function of human skeletal muscle fibres

Biopsy samples were taken from vastus lateralis muscle of seven young (YO, age 30.2 ± 2.2 years), and seven elderly (EL, age 72.7 ± 2.3 years) subjects and two elderly subjects whose right leg had been immobilized for 3.5 months (EL‐IMM, ages 70 and 75). The following main parameters were studied: (1) myosin heavy chain (MHC) isoform distribution of the samples, determined by SDS‐PAGE; (2) cross‐sectional area (CSA), specific force (Po/CSA) and maximum shortening velocity (Vo) of a large population (n= 593) of single skinned muscle fibres, classified on the basis of MHC isoform composition determined by SDS‐PAGE; (3) actin sliding velocity (Vf) on pure myosin isoforms determined by in vitro motility assays; (4) myosin concentration in single fibres determined by quantitative SDS‐PAGE. MHC isoform distribution was shifted towards fast isoforms in EL and to a larger extent in EL‐IMM. In EL and, more consistently, in EL‐IMM we observed a higher percentage of hybrid fibres than in YO, and noted the presence of MHC‐neonatal and of unusual hybrid fibres containing more than two MHC isoforms. Po/CSA significantly decreased in type 1 and 2A fibres in the order YO → EL → EL‐IMM. Vo of type 1 and 2A fibres was significantly lower in EL and higher in EL‐IMM than in YO, i.e. immobilization more than counteracted the age‐dependent decrease in Vo. The latter phenomenon was not observed for Vf. Vf on myosin 1 was lower in both EL and EL‐IMM than in YO. Vf on myosin 2X was lower in EL than in YO, and a similar trend was observed for myosin 2A. Myosin concentration decreased in type 1 and 2A fibres in the order YO → EL → EL‐IMM and was linearly related to the Po/CSA values of corresponding fibre types from the same subjects. The experiments suggest that (1) myosin concentration is a major determinant of the lower Po/CSA of single fibres in ageing and especially following immobilization and (2) ageing is associated with lower Vo of single fibres due to changes in the properties of myosin itself, whereas immobilization is associated with higher Vo in the absence of a change in myosin function.

Human circulating AC133 + stem cells restore dystrophin expression and ameliorate function in dystrophic skeletal muscle

Duchenne muscular dystrophy (DMD) is a common X-linked disease characterized by widespread muscle damage that invariably leads to paralysis and death. There is currently no therapy for this disease. Here we report that a subpopulation of circulating cells expressing AC133, a well-characterized marker of hematopoietic stem cells, also expresses early myogenic markers. Freshly isolated, circulating AC133(+) cells were induced to undergo myogenesis when cocultured with myogenic cells or exposed to Wnt-producing cells in vitro and when delivered in vivo through the arterial circulation or directly into the muscles of transgenic scid/mdx mice (which allow survival of human cells). Injected cells also localized under the basal lamina of host muscle fibers and expressed satellite cell markers such as M-cadherin and MYF5. Furthermore, functional tests of injected muscles revealed a substantial recovery of force after treatment. As these cells can be isolated from the blood, manipulated in vitro, and delivered through the circulation, they represent a possible tool for future cell therapy applications in DMD disease or other muscular dystrophies.

Unloaded shortening velocity and myosin heavy chain and alkali light chain isoform composition in rat skeletal muscle fibres.

1. This study aims to assess the role of myosin heavy chain (MHC) and alkali myosin light chain (MLC) isoforms in determining maximum velocity of shortening in fast skeletal muscle fibres. 2. The maximum velocity of shortening as determined by the slack test (Vo) was tested for its relationship with MHC composition and with alkali MLC isoform ratio of fast fibres of known MHC composition. 3. MHC isoform composition was determined using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and monoclonal antibodies against MHCs, and combining the results obtained using the two methods. Three groups of fast fibres containing only one MHC isoform were identified: IIA, IIX and IIB fibres containing respectively IIA MHC, IIX MHC and IIB MHC. Fibres containing more than one MHC isoform were discarded. 4. The mean Vo value of IIA fibres was 2.33 +/‐ 0.29 muscle lengths per second (L s‐1; mean +/‐ S.D.), this was significantly lower than that for IIX fibres (3.07 +/‐ 0.70 L s‐1) which in turn had a mean Vo value significantly lower than that for IIB fibres (3.69 +/‐ 1.01 L s‐1). 5. The relative proportion of alkali MLC isoforms (MLC3f, MLC1f) was determined by means of electrophoretic separation and densitometric quantification and was expressed as MLC3f/MLC2f with reference to the dithio‐nitrobenzoic acid (DTNB) light chain (MLC2f). The mean value of the MLC3f/MLC2f ratio was significantly lower in IIA than in IIX and IIB fibres. 6. Vo was found to be proportional to the relative content of MLC3f in IIA, IIX and IIB fibres.(ABSTRACT TRUNCATED AT 250 WORDS)

Transplantation of Genetically Corrected Human iPSC-Derived Progenitors in Mice with Limb-Girdle Muscular Dystrophy

Genetically corrected mesoangioblasts from human iPSCs derived from limb-girdle muscular dystrophy patients produce muscle fibers expressing the therapeutic gene in a mouse model of the disease. Muscle Progenitors Find Their Way Home Muscular dystrophies are genetic disorders primarily affecting skeletal muscle that result in greatly impaired mobility and, in severe cases, respiratory and cardiac dysfunction. There is no effective treatment, although several new approaches are entering clinical testing including cell therapy. Cell therapy aims to replace lost muscle fibers by transplanting healthy donor muscle progenitor cells or cells from dystrophic patients that have been genetically corrected in vitro. Mesoangioblasts are progenitor cells from blood vessel walls that have shown potential as a cell therapy in animal models of muscular dystrophy. In a new study, Tedesco et al. explore whether genetically corrected mesoangioblasts from patients with limb-girdle muscular dystrophy 2D (LGMD2D) have potential as an autologous cell therapy to treat this disease. The authors quickly found that they could not derive a sufficient number of mesoangioblasts from LGMD2D patients because the muscles of the patients were depleted of these progenitor cells. To overcome this problem, the authors reprogrammed fibroblasts or myoblasts from the LGMD2D patients to obtain human induced pluripotent stem cells (iPSCs) and induced them to differentiate into mesoangioblast-like cells that were then genetically corrected in vitro using a viral vector expressing the defective gene SGCA, which encodes α-sarcoglycan. After intramuscular or intra-arterial injection of these genetically corrected, iPSC-derived mesoangioblasts into mice with LGMD2D (immune-deficient Sgca-null mice), the cells homed to damaged mouse skeletal muscle, engrafted, and formed muscle fibers expressing α-sarcoglycan. Using mouse iPSC-derived mesoangioblasts, the researchers showed that the transplanted engrafted cells imbued muscle with greater strength and enabled the dystrophic mice to run for longer on a treadmill than dystrophic mice that did not receive the cells. This strategy offers the advantage of being able to produce unlimited numbers of genetically corrected progenitor cells, which perhaps could be used in the future as cell therapy for treating LGMD2D and other forms of muscular dystrophy. Mesoangioblasts are stem/progenitor cells derived from a subset of pericytes found in muscle that express alkaline phosphatase. They have been shown to ameliorate the disease phenotypes of different animal models of muscular dystrophy and are now undergoing clinical testing in children affected by Duchenne’s muscular dystrophy. Here, we show that patients with a related disease, limb-girdle muscular dystrophy 2D (LGMD2D), which is caused by mutations in the gene encoding α-sarcoglycan, have reduced numbers of this pericyte subset and thus produce too few mesoangioblasts for use in autologous cell therapy. Hence, we reprogrammed fibroblasts and myoblasts from LGMD2D patients to generate human induced pluripotent stem cells (iPSCs) and developed a protocol for the derivation of mesoangioblast-like cells from these iPSCs. The iPSC-derived mesoangioblasts were expanded and genetically corrected in vitro with a lentiviral vector carrying the gene encoding human α-sarcoglycan and a promoter that would ensure expression only in striated muscle. When these genetically corrected human iPSC-derived mesoangioblasts were transplanted into α-sarcoglycan–null immunodeficient mice, they generated muscle fibers that expressed α-sarcoglycan. Finally, transplantation of mouse iPSC-derived mesoangioblasts into α-sarcoglycan–null immunodeficient mice resulted in functional amelioration of the dystrophic phenotype and restoration of the depleted progenitors. These findings suggest that transplantation of genetically corrected mesoangioblast-like cells generated from iPSCs from LGMD2D patients may be useful for treating this type of muscular dystrophy and perhaps other forms of muscular dystrophy as well.

Whole-muscle and single-fibre contractile properties and myosin heavy chain isoforms in humans.

The contractile characteristics of three human muscle groups (triceps surae, quadriceps femoris and triceps brachii) of seven young male subjects were examined. The contractile properties were determined from electrically evoked isometric responses and compared with fibre type composition determined from needle biopsy samples. Fibre types were identified using myosin heavy chain (MHC) isoforms as molecular markers with gel electrophoresis (SDS-PAGE) and histochemical ATPase staining. Four contractile parameters (twitch time to peak torque, the maximal rate of torque development, frequency response and fatiguability) were found to be related to fibre type composition. From the biopsy samples, single muscle fibres were isolated and chemically skinned. Isometric tension (Po) unloaded shortening velocity (Vo) and rate of tension rise (dP/dt) were determined. Each fibre was classified on the basis of its MHC isoform composition determined by SDS-PAGE. Fibres belonging to the same type showed identical contractile parameters regardless of the muscle of origin, except minor differences in Po of the fast fibres and dP/dt of slow fibres. The results are in favour of the conclusion that fibre type composition, determined using MHC isoforms as markers, is the major determinant of the diversity of contractile properties among human muscle groups.

Branched-Chain Amino Acid Supplementation Promotes Survival and Supports Cardiac and Skeletal Muscle Mitochondrial Biogenesis in Middle-Aged Mice

Recent evidence points to a strong relationship between increased mitochondrial biogenesis and increased survival in eukaryotes. Branched-chain amino acids (BCAAs) have been shown to extend chronological life span in yeast. However, the role of these amino acids in mitochondrial biogenesis and longevity in mammals is unknown. Here, we show that a BCAA-enriched mixture (BCAAem) increased the average life span of mice. BCAAem supplementation increased mitochondrial biogenesis and sirtuin 1 expression in primary cardiac and skeletal myocytes and in cardiac and skeletal muscle, but not in adipose tissue and liver of middle-aged mice, and this was accompanied by enhanced physical endurance. Moreover, the reactive oxygen species (ROS) defense system genes were upregulated, and ROS production was reduced by BCAAem supplementation. All of the BCAAem-mediated effects were strongly attenuated in endothelial nitric oxide synthase null mutant mice. These data reveal an important antiaging role of BCAAs mediated by mitochondrial biogenesis in mammals.