Altered skeletal muscle mitochondrial biogenesis but improved endurance capacity in trained OPA1-deficient mice.

The role of OPA1, a GTPase dynamin protein mainly involved in the fusion of inner mitochondrial membranes, has been studied in many cell types, but only a few studies have been conducted on adult differentiated tissues such as cardiac or skeletal muscle cells. Yet OPA1 is highly expressed in these cells, and could play different roles, especially in response to an environmental stress like exercise. Endurance exercise increases energy demand in skeletal muscle and repeated activity induces mitochondrial biogenesis and activation of fusion-fission cycles for the synthesis of new mitochondria. But currently no study has clearly shown a link between mitochondrial dynamics and biogenesis. Using a mouse model of haploinsufficiency for the Opa1 gene (Opa1(+/-)), we therefore studied the impact of OPA1 deficiency on the adaptation ability of fast skeletal muscles to endurance exercise training. Our results show that, surprisingly, Opa1(+/-) mice were able to perform the same physical activity as control mice. However, the adaptation strategies of both strains after training differed: while in control mice mitochondrial biogenesis was increased as expected, in Opa1(+/-) mice this process was blunted. Instead, training in Opa1(+/-) mice led to an increase in endurance capacity, and a specific adaptive response involving a metabolic remodelling towards enhanced fatty acid utilization. In conclusion, OPA1 appears necessary for the normal adaptive response and mitochondrial biogenesis of skeletal muscle to training. This work opens new perspectives on the role of mitochondrial dynamics in skeletal muscle cells and during adaptation to stress.

Structure and composition of tubular aggregates of skeletal muscle fibres.

Unusual regions of densely packed membranous tubules known as tubular aggregates (TAs) have been observed in skeletal muscle fibres of mammals under numerous pathological conditions but also in health. Their causality is unclear. It is neither known whether TAs are destructive and should be treated or whether they have a compensating function in an endangered muscle. In spite of many similarities, the histochemical, immunocytochemical and ultrastructural characteristics of tubular aggregates do vary. Histochemistry provided an overall characteristic of TAs as membranous inclusions with a variety of enzymatic activities. Immunocytochemical evidence revealed that tubular aggregates contain miscellaneous proteins and that derive from membranes of sarcoplasmic reticulum and mitochondria. No evidence for the presence of contractile and cytoskeletal proteins in TAs was found. Ultrastructurally, TAs are characterized as more or less densely packed aggregates of vesicular or tubular membranes of variable forms and sizes that may contain amorphous material, filaments or inner tubules. Various reported types of tubular aggregates, namely, proliferating terminal cisterns, vesicular membrane collections, TAs with double-walled tubules, TAs with single-walled tubules, aggregates of dilated tubules with inner tubules, aggregates of tubulo-filamentous structures, filamentous tubules, riesentubuli, and related membranous structures including cylindrical spirals are sumarized and analyzed here in detail.

Energetic crosstalk between organelles: architectural integration of energy production and utilization.

Cells with high and fluctuating energy demands such as cardiomyocytes need efficient systems to link energy production to energy utilization. This is achieved in part by compartmentalized energy transfer enzymes such as creatine kinase (CK). However, hearts from CK-deficient mice develop normal cardiac function under conditions of moderate workload. We have therefore investigated whether a direct functional interplay exists between mitochondria and sarcoplasmic reticulum or between mitochondria and myofilaments in cardiac cells that catalyzes direct energy and signal channeling between organelles. We used the selective permeabilization of sarcolemmal membranes with saponin to study the functional interactions between organelles within the cellular architecture. We measured contractile kinetics, oxygen consumption, and caffeine-induced tension transients. The results show that in hearts of normal mice, ATP produced by mitochondria (supplied with substrates, oxygen, and adenine nucleotides) was able to sustain calcium uptake and contractile speed. Moreover, direct mitochondrially supplied ATP was nearly as effective as CK-supplied ATP and much more effective than externally supplied ATP, suggesting that a direct ATP/ADP channeling exists between the sites of energy production (mitochondria) and energy utilization (sarcoplasmic reticulum and myofilaments). On the other hand, in cardiac cells of mice deficient in mitochondrial and cytosolic CK, marked cytoarchitectural modifications were observed, and direct adenine nucleotide channeling between mitochondria and organelles was still effective for sarcoplasmic reticulum and myofilaments. Such direct crosstalk between organelles may explain the preserved cardiac function of CK-deficient mice under moderate workloads.

Ultrastructure and innervation of regenerated intrafusal muscle fibres in heterochronous isografts of the fast rat muscle.

Extensor digitorum longus (EDL) muscles from 2- to 28-day-old rats were grafted into EDL muscles of adult inbred recipients (n = 8). At 1-6 months after the operation, experimental muscles were excised and the ultrastructure and innervation of regenerated muscle spindles was examined. Regenerated muscle spindles (n = 36) in isografted EDL muscles contained 4.3 +/- 0.2 (mean +/- SEM) encapsulated muscle fibres. These "intrafusal" muscle fibres lacked nuclear bag and nuclear chain accumulations, which are characteristic of normal muscle spindles; thus, they rather resembled thin encapsulated extrafusal muscle fibres. In the same sample, myelinated axons were found in 33 (92%) muscle spindles, but no sensory terminals were found. These findings demonstrate that regenerated spindles in isografted EDL muscles were not reinnervated by spindle-specific sensory axons, but exclusively by motor axons. Typical intracapsular motor endplates (MEPs) were found in one third of regenerated spindles examined. Their motor terminals contained accumulated mitochondria and synaptic vesicles. As is characteristic for MEPs, axolemma and sarcolemma were separated by a synaptic cleft about 60 nm wide that contained a basal lamina. The underlying sarcolemma formed either small infoldings or none at all, and the subsynaptic area contained only small subsarcolemmal accumulations of mitochondria. It is apparent that the structures described here as "regenerated muscle spindles" do not perform their normal physiological function as stretch receptors because they lack the sensory innervation. The present results show that regeneration and reinnervation in heterochronous isografts corresponds to that previously described in autotransplanted free muscle grafts. The results also show that, during muscle spindle regeneration, intrafusal satellite cells develop into extrafusal-like muscle fibres, apparently due to their motor innervation.

Alternative strategies in muscle genotype and phenotype studies. A model of intrafusal muscle fibre type differentiation.

The development and regeneration of muscle fibres start from myoblasts of embryos or adult animals. The resulting phenotype is a combination of genetically fixed properties of myoblast cell lineages and of extrinsic, primarily neurogenic factors. Intrafusal fibre types of muscle spindles differ from each other and from extrafusal fibres by their ultrastructure, by the presence of both sensory and motor innervation, and by the content of specific myosin heavy chain (MHC) isoforms. Differentiation of these distinctions depends on the morphogenetic influence of primary afferent neurones. It is, however, not known, whether the intrafusal phenotype can be induced in any myotube regardless of its cell line origin or only in a special predetermined intrafusal lineage(s) committed to differentiate into intrafusal muscle fibres. The aim of our studies was to define the contribution of intrinsic myogenic properties of muscle cell lineage and extrinsic neurogenic factors by the sensory and the motor innervation on the differentiation of intrafusal phenotypes using ultrastructural analysis and immunocytochemical determination of MHCs under various experimental conditions. The presented minireview is based on the results of our previous findings, and preliminary experiments indicate that new important results may be obtained in studies of myogenesis and muscle regeneration.

Neomyogenesis in neonatally de-efferented and postnatally denervated rat muscle spindles.

The ultrastructure of muscle spindles de-efferented by the extirpation of the lumbosacral spinal cord at the age of 2 days and subsequently deprived of their sensory innervation by the section of the sciatic nerve at 3-4 weeks of age was studied in serial sections of 2-month-old rat hindlimb muscles. De-efferentation leaves the primary sensory neurons and their peripheral axons intact and capable of inducing the muscle spindle morphogenesis during the critical period of their development. In de-efferented and subsequently denervated muscle spindles, new supernumerary intrafusal muscle profiles (SIPs) appeared in the muscle spindle A region. They were formed in intimate spatial relation with the original intrafusal muscle fibres (IMFs) predominantly from activated satellite cells derived from both nuclear bag (larger diameter) and nuclear chain fibres. SIPs, however, lacked the typical nuclear accumulations, as well as other ultrastructural distinctions present in control IMFs. The majority of differentiated SIPs separated from original IMFs, whereas the less differentiated SIPs were usually closely apposed to the surface of the parent IMFs and both were covered by the common basal lamina. In some spindles, the original IMFs and/or new SIPs at different stages of their differentiation were found together and they formed clusters of variable shape and composition. In the majority of clusters, all profiles seemed to be isolated along their entire length, although in few clusters, occasional cytoplasmic connections of variable length between intrafusal profiles were found. This result is important for the interpretation of the forthcoming study of expression of muscle spindle-specific myosin heavy chain isoforms in denervated SIPs in rat muscle spindles gradually deprived of their motor and sensory innervation.

Structural characteristics and distribution of satellite cells along crayfish muscle fibers.

The distribution of satellite cells (sc) in long-sarcomere muscle fibers from the carpopod extensor muscle of the crayfish (Astacus fluviatilis) has been studied electron-microscopically. The sc are spindle-shaped and are oriented parallel to the long axis of a fiber. The mean lengths of sc nuclei (17.00 microns) and that of myonuclei (18.35 microns) differ non-significantly. In older animals, the mean ratio of the number of sc nuclei to the total number of nuclei (sc nuclei + myonuclei) is 0.0716, 0.0848, and 0.034 for the tendon, central and shell segments, respectively. The corresponding values for younger animals are 0.158, 0.166, and 0.081. The mean numbers of sc nuclei per mm of a fiber are 94, 117, and 47 (older animals), and 164, 117, and 94 (younger animals) for the tendon, central and shell segments, respectively. The high incidence of sc per unit fiber length in crayfish may be related to the fact that crayfish muscle fibers have a much larger diameter than vertebrate muscle cells.

Ryanodine receptor purified from crayfish skeletal muscle.

The ryanodine receptor was isolated from the sarcoplasmic reticulum of crayfish skeletal muscle. Ryanodine binding to the native fraction was measured by Scatchard analysis and values of 60 nmol/l and 9 pmol/mg were obtained for KD and Bmax respectively. The identity of purified receptor was confirmed by electron microscopy, electrophoresis and incorporation into planar lipid bilayers. At least two conductance states (100 pS and 50 pS) were observed in 100 mmol/l NaCl both for native and purified receptor.

Na(+)-Ca2+ exchange in locust striated muscles.

High Na+ + Ca2+ exchange rates comparable with those reported for crayfish striated muscle, rat heart and rat brain, were observed in locust striated muscle homogenates and membrane preparations. The Na(+)-Ca2+ exchange followed the 1st order kinetics with a Km value of 18 mumol.l-1 for Ca, the pH optimum was at 8, the temperature optimum at 30 degrees C, and the exchange was inhibited in the presence of sodium in the incubation medium, with a KiNa of approx. 25 mmol.l-1. The present results suggest a high Na(+)-Ca2+ exchange in locust striated muscles which operate on the calcium electrogenesis principle.

Characterization of DHP binding protein in crayfish striated muscle.

The dihydropyridine calcium channel blocker, [3H]PN 200-110, binds specifically also to crayfish muscle membranes, though with a binding capacity smaller than that measured with rabbit or human skeletal muscle membranes. [3H]PN 200-110 binding proteins from the crayfish T-tubules were solubilized and purified on WGA Sepharose or extracted from gel. The purified protein has a molecular mass of approximately 190 kDa under nonreducing conditions and was able to transport calcium after reconstitution. Polyclonal antibodies against crayfish T-tubules enriched with purified DHP-binding protein were shown to bind to DHP-binding protein from both the crayfish and the rabbit skeletal muscle, although not with the same intensity. Electron microscopy showed the presence of ovoid particles. Our results suggest that a voltage-dependent calcium channel may be present in crayfish skeletal muscle, which is homological with the L-type calcium channel in rabbit skeletal muscle.

Arrangement and density of junctional feet in crayfish muscle fibres.

Junctional feet in tubulo-reticular junctions of crayfish muscle fibres are arranged tetragonally with a centre-to-centre spacing of 30-34 nm. The resulting density of 860-1110 feet per 1 micron 2 of the junctional membrane is similar to that reported for other animal species. Using data of a previous stereological study, there are 150-190 feet per 1 micron 2 of the total T-tubule surface and 6000-7800 feet per 100 microns 3 of the fibre volume.

[Slow asymmetric currents and ultrastructure of tubulo-reticular contacts in the muscle fibers of the crayfish]. / Medlennye asimmetrichnye toki i ul'trastruktura tubulo-retikuliarnykh kontaktov v myshechnykh voloknakh raka.

Asymmetric membrane currents were studied in isolated muscle fibres of the crayfish Astacus fluviatilis under conditions of voltage clamp and controlled composition of both the external and internal environment. In addition to fast asymmetric currents, probably associated with the opening of calcium channels in the superficial membrane, slow asymmetric currents could be also observed in this type of muscle fibre; the time course of the latter currents was almost by one order slower than that of the fast muscle fibres of the frog. The value of the displaced charge was related to the number of feet in the tubulo-reticular (TR) junction. The number of feet and their distribution in the TR junction were studied using transmission electron microscopy. The amount of charges displaced per one foot supports the hypothesis stating that slow displacements currents are associated with displacement of charged particles distributed over the entire area of sarcolemmal and tubular invaginations rather than in the feet alone.

A quantitative estimation of components in crayfish muscle fibres by stereological methods.

The volume and surface densities of structural components in isolated long-sarcomere fibres of crayfish (Astacus fluviatilis) were estimated by stereological methods. The relative volumes were as follows (in % of the fibre volume): sarcolemmal invaginations (SI) 0.74; T-system 1.1; whole sarcoplasmic reticulum (SR) 4.7; junctional SR cisternae 0.41; mitochondria 1.1; nuclei 0.57; myofibrils 81.6; peripheral basal lamina 1.1; junctional gap 0.043; lysosomes 0.029. The values for surface densities were (in microns2/100 microns3): peripheral sarcolemma 1.27; SI 7.21; T-system 40.2; whole SR 146.4; junctional SR cisternae 14.88; junctional area of SR cisternae 7.05; mitochondria 8.98; nuclei 0.75; myofibrils 163. The invaginated membranes (SI and T-system) made up 97.4% of the total area of surface membranes and increased the effective area of peripheral sarcolemma 39 times. Using the values of the total electrical capacity from previous investigations on Astacus fibres, the specific capacity of surface membranes was calculated as 1.08 microF/cm2.