Simona Boncompagni

The contribution of reactive oxygen species to sarcopenia and muscle ageing

In recent years, age-related diseases and disabilities have become of major interest and importance for health. This holds particularly for the Western community, where the remarkable improvement of medical health, standard of living, and hygiene have reduced the main causes of death. Despite numerous theories and intensive research, the principal molecular mechanisms underlying the process of aging are still unknown. Most, if not all, attempts to prevent or stop the onset of typical degenerative diseases associated with aging have so far been futile. Solutions to the major problems of dealing with age-related diseases can only come from a systematic and thorough molecular analysis of the aging process and a detailed understanding of its causes.

RyR1 S-Nitrosylation Underlies Environmental Heat Stroke and Sudden Death in Y522S RyR1 Knockin Mice

Mice with a malignant hyperthermia mutation (Y522S) in the ryanodine receptor (RyR1) display muscle contractures, rhabdomyolysis, and death in response to elevated environmental temperatures. We demonstrate that this mutation in RyR1 causes Ca(2+) leak, which drives increased generation of reactive nitrogen species (RNS). Subsequent S-nitrosylation of the mutant RyR1 increases its temperature sensitivity for activation, producing muscle contractures upon exposure to elevated temperatures. The Y522S mutation in humans is associated with central core disease. Many mitochondria in the muscle of heterozygous Y522S mice are swollen and misshapen. The mutant muscle displays decreased force production and increased mitochondrial lipid peroxidation with aging. Chronic treatment with N-acetylcysteine protects against mitochondrial oxidative damage and the decline in force generation. We propose a feed-forward cyclic mechanism that increases the temperature sensitivity of RyR1 activation and underlies heat stroke and sudden death. The cycle eventually produces a myopathy with damaged mitochondria.

Mitochondria are linked to calcium stores in striated muscle by developmentally regulated tethering structures.

Bi-directional calcium (Ca(2+)) signaling between mitochondria and intracellular stores (endoplasmic/sarcoplasmic reticulum) underlies important cellular functions, including oxidative ATP production. In striated muscle, this coupling is achieved by mitochondria being located adjacent to Ca(2+) stores (sarcoplasmic reticulum [SR]) and in proximity of release sites (Ca(2+) release units [CRUs]). However, limited information is available with regard to the mechanisms of mitochondrial-SR coupling. Using electron microscopy and electron tomography, we identified small bridges, or tethers, that link the outer mitochondrial membrane to the intracellular Ca(2+) stores of muscle. This association is sufficiently strong that treatment with hypotonic solution results in stretching of the SR membrane in correspondence of tethers. We also show that the association of mitochondria to the SR is 1) developmentally regulated, 2) involves a progressive shift from a longitudinal clustering at birth to a specific CRU-coupled transversal orientation in adult, and 3) results in a change in the mitochondrial polarization state, as shown by confocal imaging after JC1 staining. Our results suggest that tethers 1) establish and maintain SR-mitochondrial association during postnatal maturation and in adult muscle and 2) likely provide a structural framework for bi-directional signaling between the two organelles in striated muscle.

Reorganized stores and impaired calcium handling in skeletal muscle of mice lacking calsequestrin-1

Calsequestrin (CS), the major Ca2+‐binding protein in the sarcoplasmic reticulum (SR), is thought to play a dual role in excitation–contraction coupling: buffering free Ca2+ increasing SR capacity, and modulating the activity of the Ca2+ release channels (RyRs). In this study, we generated and characterized the first murine model lacking the skeletal CS isoform (CS1). CS1‐null mice are viable and fertile, even though skeletal muscles appear slightly atrophic compared to the control mice. No compensatory increase of the cardiac isoform CS2 is detectable in any type of skeletal muscle. CS1‐null muscle fibres are characterized by structural and functional changes, which are much more evident in fast‐twitch muscles (EDL) in which most fibres express only CS1, than in slow‐twitch muscles (soleus), where CS2 is expressed in about 50% of the fibres. In isolated EDL muscle, force development is preserved, but characterized by prolonged time‐to‐peak and half‐relaxation time, probably related to impaired calcium release from and re‐uptake by the SR. Ca2+‐imaging studies show that the amount of Ca2+ released from the SR and the amplitude of the Ca2+ transient are significantly reduced. The lack of CS1 also causes significant ultrastructural changes, which include: (i) striking proliferation of SR junctional domains; (ii) increased density of Ca2+‐release channels (confirmed also by 3H‐ryanodine binding); (iii) decreased SR terminal cisternae volume; (iv) higher density of mitochondria. Taken together these results demonstrate that CS1 is essential for the normal development of the SR and its calcium release units and for the storage and release of appropriate amounts of SR Ca2+.

Unexpected Structural and Functional Consequences of the R33Q Homozygous Mutation in Cardiac Calsequestrin: A Complex Arrhythmogenic Cascade in a Knock In Mouse Model

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disorder characterized by life threatening arrhythmias elicited by physical and emotional stress in young individuals. The recessive form of CPVT is associated with mutation in the cardiac calsequestrin gene (CASQ2). We engineered and characterized a homozygous CASQ2R33Q/R33Q mouse model that closely mimics the clinical phenotype of CPVT patients. CASQ2R33Q/R33Q mice develop bidirectional VT on exposure to environmental stress whereas CASQ2R33Q/R33Q myocytes show reduction of the sarcoplasmic reticulum (SR) calcium content, adrenergically mediated delayed (DADs) and early (EADs) afterdepolarizations leading to triggered activity. Furthermore triadin, junctin, and CASQ2-R33Q proteins are significantly decreased in knock-in mice despite normal levels of mRNA, whereas the ryanodine receptor (RyR2), calreticulin, phospholamban, and SERCA2a-ATPase are not changed. Trypsin digestion studies show increased susceptibility to proteolysis of mutant CASQ2. Despite normal histology, CASQ2R33Q/R33Q hearts display ultrastructural changes such as disarray of junctional electron-dense material, referable to CASQ2 polymers, dilatation of junctional SR, yet normal total SR volume. Based on the foregoings, we propose that the phenotype of the CASQ2R33Q/R33Q CPVT mouse model is portrayed by an unexpected set of abnormalities including (1) reduced CASQ2 content, possibly attributable to increased degradation of CASQ2-R33Q, (2) reduction of SR calcium content, (3) dilatation of junctional SR, and (4) impaired clustering of mutant CASQ2.

Lifelong Physical Exercise Delays Age-Associated Skeletal Muscle Decline

Aging is usually accompanied by a significant reduction in muscle mass and force. To determine the relative contribution of inactivity and aging per se to this decay, we compared muscle function and structure in (a) male participants belonging to a group of well-trained seniors (average of 70 years) who exercised regularly in their previous 30 years and (b) age-matched healthy sedentary seniors with (c) active young men (average of 27 years). The results collected show that relative to their sedentary cohorts, muscle from senior sportsmen have: (a) greater maximal isometric force and function, (b) better preserved fiber morphology and ultrastructure of intracellular organelles involved in Ca(2+) handling and ATP production, (c) preserved muscle fibers size resulting from fiber rescue by reinnervation, and (d) lowered expression of genes related to autophagy and reactive oxygen species detoxification. All together, our results indicate that: (a) skeletal muscle of senior sportsmen is actually more similar to that of adults than to that of age-matched sedentaries and (b) signaling pathways controlling muscle mass and metabolism are differently modulated in senior sportsmen to guarantee maintenance of skeletal muscle structure, function, bioenergetic characteristics, and phenotype. Thus, regular physical activity is a good strategy to attenuate age-related general decay of muscle structure and function (ClinicalTrials.gov: NCT01679977).

Structural differentiation of skeletal muscle fibers in the absence of innervation in humans

The relative importance of muscle activity versus neurotrophic factors in the maintenance of muscle differentiation has been greatly debated. Muscle biopsies from spinal cord injury patients, who were trained with an innovative protocol of functional electrical stimulation (FES) for prolonged periods (2.4–9.3 years), offered the unique opportunity of studying the structural recovery of denervated fibers from severe atrophy under the sole influence of muscle activity. FES stimulation induced surprising recovery of muscle structure, mass, and force even in patients whose muscles had been denervated for prolonged periods before the beginning of FES training (up to 2 years) and had almost completely lost muscle-specific internal organization. Ninety percent (or more) of the fibers analyzed by electron microscopy showed a striking recovery of the ultrastructural organization of myofibrils and Ca2+-handling membrane systems. This functional/structural restoration follows a pattern that mimics some aspects of normal muscle differentiation. Most importantly, the recovery occurs in the complete absence of motor and sensory innervation and of nerve-derived trophic factors, that is, solely under the influence of muscle activity induced by electrical stimulation.

Home-based functional electrical stimulation rescues permanently denervated muscles in paraplegic patients with complete lower motor neuron lesion

Background. Spinal cord injury causes muscle wasting and loss of function, which are especially severe after complete and permanent damage to lower motor neurons. In a previous cross-sectional study, long-standing denervated muscles were rescued by home-based functional electrical stimulation (h-bFES) training. Objective. To confirm results by a 2-year longitudinal prospective study of 25 patients with complete conus/cauda equina lesions. Methods. Denervated leg muscles were stimulated by h-bFES using a custom-designed stimulator and large surface electrodes. Muscle mass, force, and structure were determined before and after 2 years of h-bFES using computed tomography, measurements of knee torque during stimulation, and muscle biopsies analyzed by histology and electron microscopy. Results. Twenty of 25 patients completed the 2-year h-bFES program, which resulted in (a) a 35% cross-sectional increase in area of the quadriceps muscle from 28.2 ± 8.1 to 38.1 ± 12.7 cm 2 (P < .001), a 75% increase in mean diameter of muscle fibers from 16.6 ± 14.3 to 29.1 ± 23.3 μm (P < .001), and improvements of the ultrastructural organization of contractile material; and (b) a 1187% increase in force output during electrical stimulation from 0.8 ± 1.3 to 10.3 ± 8.1 N m (P < .001). The recovery of quadriceps force was sufficient to allow 25% of the subjects to perform FES-assisted stand-up exercises. Conclusions. Home-based FES of denervated muscle is an effective home therapy that results in rescue of muscle mass and tetanic contractility. Important immediate benefits for the patients are the improved cosmetic appearance of lower extremities and the enhanced cushioning effect for seating.

Mitochondrial superoxide flashes: metabolic biomarkers of skeletal muscle activity and disease

Mitochondrial superoxide flashes (mSOFs) are stochastic events of quantal mitochondrial superoxide generation. Here, we used flexor digitorum brevis muscle fibers from transgenic mice with muscle‐specific expression of a novel mitochondrial‐targeted superoxide biosensor (mt‐cpYFP) to characterize mSOF activity in skeletal muscle at rest, following intense activity, and under pathological conditions. Results demonstrate that mSOF activity in muscle depended on electron transport chain and adenine nucleotide translocase functionality, but it was independent of cyclophilin‐D‐mediated mitochondrial permeability transition pore activity. The diverse spatial dimensions of individual mSOF events were found to reflect a complex underlying morphology of the mitochondrial network, as examined by electron microscopy. Muscle activity regulated mSOF activity in a biphasic manner. Specifically, mSOF frequency was significantly increased following brief tetanic stimulation (18.1 ± 1.6 to 22.3±2.0 flashes/1000 μm2·100 s before and after 5 tetani) and markedly decreased (to 7.7±1.6 flashes/1000 μm2 ·100 s) following prolonged tetanic stimulation (40 tetani). A significant temperature‐dependent increase in mSOF frequency (11.9±0.8 and 19.8±2.6 flashes/1000 μm2 ·100 s at 23°C and 37°C) was observed in fibers from RYR1Y522S/WT mice, a mouse model of malignant hyperthermia and heat‐induced hypermetabolism. Together, these results demonstrate that mSOF activity is a highly sensitive biomarker of mitochondrial respiration and the cellular metabolic state of muscle during physiological activity and pathological oxidative stress.—Wei, L., Salahura, G., Boncompagni, S., Kasischke, K. A., Protasi, F., Sheu, S.‐S., Dirksen, R. T. Mitochondrial superoxide flashes: metabolic biomarkers of skeletal muscle activity and disease. FASEB J. 25, 3068–3078 (2011). www.fasebj.org

Characterization and temporal development of cores in a mouse model of malignant hyperthermia

Malignant hyperthermia (MH) and central core disease are related skeletal muscle diseases often linked to mutations in the type 1 ryanodine receptor (RYR1) gene, encoding for the Ca2+ release channel of the sarcoplasmic reticulum (SR). In humans, the Y522S RYR1 mutation is associated with malignant hyperthermia susceptibility (MHS) and the presence in skeletal muscle fibers of core regions that lack mitochondria. In heterozygous Y522S knock-in mice (RYR1Y522S/WT), the mutation causes SR Ca2+ leak and MHS. Here, we identified mitochondrial-deficient core regions in skeletal muscle fibers from RYR1Y522S/WT knock-in mice and characterized the structural and temporal aspects involved in their formation. Mitochondrial swelling/disruption, the initial detectable structural change observed in young-adult RYR1Y522S/WT mice (2 months), does not occur randomly but rather is confined to discrete areas termed presumptive cores. This localized mitochondrial damage is followed by local disruption/loss of nearby SR and transverse tubules, resulting in early cores (2–4 months) and small contracture cores characterized by extreme sarcomere shortening and lack of mitochondria. At later stages (1 year), contracture cores are extended, frequent, and accompanied by areas in which contractile elements are also severely compromised (unstructured cores). Based on these observations, we propose a possible series of events leading to core formation in skeletal muscle fibers of RYR1Y522S/WT mice: Initial mitochondrial/SR disruption in confined areas causes significant loss of local Ca2+ sequestration that eventually results in the formation of contractures and progressive degradation of the contractile elements.