Immunoelectron microscopic epitope locations of titin in rabbit heart muscle.

The location of cardiac titin epitopes in the sarcomere of rabbit cardiac, atrial and ventricular muscle was studied by using polyclonal antibodies against skeletal muscle titin. The results show that incubation with the antibody leads to the appearance of four electron-dense stripes in the A band of both atrial and ventricular cardiac muscle. The location and intensity of these stripes were identical to those observed in skeletal muscle. In conclusion we demonstrate that titins from skeletal and cardiac muscles share some common antigenic determinants.

Morphological and functional characterization of the endosarcomeric elastic filament.

The elastic filament was studied in chemically skinned fibers from rabbit psoas muscle by electron microscopy and resting tension measurements. Extraction of skinned fibers with 40 mM sodium pyrophosphate caused a selective removal of about two-thirds of the thick filaments and formed a gap between the remaining portion of the A band and the I band. Very thin filaments were seen in the gap and were decorated by anti-titin antibody. The resting tension of these fibers was comparable to that of unextracted control fibers. When the M band was completely extracted by a solution containing 0.6 M NaCl, the resting tension completely disappeared at sarcomere lengths from 2.8 to approximately 3.4 microns. These results suggest that the elastic force of short sarcomeres is endowed in the titin filaments and that these filaments are anchored to some structures of the Z and M lines. Other filaments were found in the gap between the two I bands of NaCl-extracted sarcomeres. These filaments differed from titin filaments by a larger diameter and the anchoring points. They may represent the sarcomeric structures responsible for the resting tension of extracted fibers stretched at sarcomere lengths longer than 3.4 microns.

The organization of titin (connectin) and nebulin in the sarcomeres: an immunocytolocalization study.

Monospecific polyclonal antibodies against two exceptionally large proteins, titin (a-T) and nebulin (a-N) isolated from rabbit skeletal muscles, were raised in guinea pig. Using an immuno-pre-embedding method, we have localized at the ultrastructural level of resolution the reactivity sites in skinned muscle fibres. At resting length a-T and a-N antibodies recognize epitopes which only partially overlap. a-T antibodies decorate mostly the A band with at least four clearly distinguished lines of reaction and one line in the I band, all near the A/I limit; a-N antibodies bind to the same region, but with wider areas of reaction in both A and I bands. To study whether the localization of these reaction sites varies according to the sarcomere length, skinned rabbit psoas fibres were incubated at sarcomere lengths ranging from maximum shortening to overstretching. The results indicate that lines decorated by a-T move away from the Z disc when the sarcomere is lengthened. With respect to the M line, the behaviour was biphasic. When the sarcomere was stretched up to about 2.8 microns, the decorated lines maintain almost the same distance from the M line. When the sarcomere is stretched beyond 2.8 microns, all a-T epitopes move away from the M line and the molecule behaves elastically. At resting length the a-N decoration appears to be localized on three large adjacent bands at the I, A/I and A level. The a-N line of reaction at the edge of the A band moves away from the Z discs as the sarcomere lengthens, while a second line which seems to be localized at the tip of the thin filament moves away from M line when the sarcomere lengthens. In non-overlapping sarcomeres a-N antibodies decorate only the tip of the thin filaments. Our results indicate that titin forms a polar filament connecting the M line to the Z line. In short sarcomeres, the filament seems to have some connections with structures of the A band, since titin epitopes do not move during stretching. These connections are lost at longer sarcomere lengths. On the other hand, our results suggest that nebulin is probably not a constituent of the titin filament.

Tubular aggregates: sarcoplasmic reticulum origin, calcium storage ability, and functional implications.

Muscle biopsy specimens from three patients with an autosomal dominant myopathy and tubular aggregates in both type 1 and type 2 fibers were investigated for immunofluorescent staining with antibodies to sarcoplasmic reticulum (SR) Ca-pump protein and calsequestrin and for Ca2+ loading ability. The results show that type 1 and type 2 fibers are differentially reactive to anti-Ca-pump protein IgG and similarly reactive with affinity-purified antibody to calsequestrin, which is in agreement with earlier observations in rat skeletal muscle. Tubular aggregates, which are shown to be highly reactive with either kind of antibody, appear to be sites of calcium accumulation for oxalate-facilitated adenosine triphosphate (ATP) dependent Ca uptake by chemically skinned fibers and thereby increase markedly the Ca loading capacity of the affected fibers.

Familial neuromuscular disease with tubular aggregates.

We have studied four members of a family in which five individuals of both sexes in three generations have mild weakness and muscle aching. Serum creatine kinase (CK) and/or aldolase were mildly to moderately elevated. Tubular aggregates were present in all patients in all fiber types. Type 1 fiber predominance and type 2 hypotrophy or atrophy was noted in three biopsies. Fiber splitting was prominent in two cases. Electron microscopic studies confirm that the tubular aggregates originate from muscle sarcoplasmic reticulum (SR). The presence of T tubules making contacts with some tubules inside the collection of tubular aggregates is suggestive of their derivation from terminal cisternae.

"Fast" isomyosins and fiber types in mammalian skeletal muscle.

Immunohistochemical procedures have been used to correlate antigenic differences among fast isomyosins with their specific localization in particular types of rat muscle fibers. Antisera were produced in rabbits against myosins isolated from guinea pig fast-white (tensor fasciae latae) and fast-red (masseter) muscles. After sequential cross-adsorptions on insolubilized heterologous myosins, antibodies mainly directed against heavy chain determinants were obtained. In indirect immunofluorescence assays, these antibodies selectively stained fast-white (type IIB) and fast-red (type IIA) fibers, respectively. Slow-twitch (type I) fibers, which were reactive with antibodies against soleus muscle myosin, were unreactive with anti-fast-myocin antibodies. In addition to these major types of fibers, with unique immunoreactivity, two groups of fibers with double reactivity were identified: 1) fibers reactive with anti-slow-twitch and anti-fast-red antibodies (corresponding to IIC fibers with enzyme histochemistry) and 2) fibers reactive with anti-fast-red and anti-fast-white antibodies. We propose to identify these fibers with the symbols I in equilibrium with IIA and IIA in equilibrium with IIB, respectively, as they may be considered as intermediate and/or transitional stages between the major fiber types. No fibers were reactive with both anti-slow-twitch and anti-fast-white antibodies. Therefore, muscle fibers may change in a sequential manner, from I leads to IIA leads to IIB or in the reverse direction, suggesting an obligatory sequence of gene activation.

Carnitine palmityl transferase deficiency: clinical variability, carrier detection, and autosomal-recessive inheritance.

A 21-year-old man had recurrent myoglobinuria; his 28-year-old sister had symptoms of fatigability. During prolonged fasting, serum free fatty acid rose in both siblings, but only the sister produced ketone bodies and had elevated creatine phosphokinase activity. Carnitine palmityl transferase (CPT) activity was less than 30% of normal in muscle and platelets. Liver biopsy disclosed a low level of the enzyme in the brother. The parents had intermediate levels of the enzyme in platelets. CPT deficiency seems to have an autosomal-recessive pattern of inheritance and a variable phenotypic expression.

Myosin types and fiber types in cardiac muscle. I. Ventricular myocardium.

Antisera against bovine atrial myosin were raised in rabbits, purified by affinity chromatography, and absorbed with insolubilized ventricular myosin. Specific anti-bovine atrial myosin (anti-bAm) antibodies reacted selectively with atrial myosin heavy chains, as determined by enzyme immunoassay combined with SDS-gel electrophoresis. In direct and indirect immunofluorescence assay, anti-bAm was found to stain all atrial muscle fibers and a minor proportion of ventricular muscle fibers in the right ventricle of the bovine heart. In contrast, almost all muscle fibers in the left ventricle were unreactive. Purkinje fibers showed variable reactivity. In the rabbit heart, all atrial muscle fibers were stained by anti-bAm, whereas ventricular fibers showed a variable response in both the right and left ventricle, with a tendency for reactive fibers to be more numerous in the right ventricle and in subepicardial regions. Diversification of fiber types with respect to anti-bAm reactivity was found to occur during late stages of postnatal development in the rabbit heart and to be influenced by thyroid hormone. All ventricular muscle fibers became strongly reactive after thyroxine treatment, whereas they became unreactive or poorly reactive after propylthiouracil treatment. These findings are consistent with the existence of different ventricular isomyosins whose relative proportions can vary according to the thyroid state. Variations in ventricular isomyosin composition can account for the changes in myosin Ca2+-activated ATPase activity previously observed in cardiac muscle from hyper- and hypothyroid animals and may be responsible for the changes in the velocity of contraction of ventricular myocardium that occur under these conditions. The differential distribution of ventricular isomyosins in the normal heart suggests that fiber types with different contractile properties may coexist in the ventricular myocardium.

Peroneal muscular atrophy with ataxia and partial myoclonic epilepsy.

Two brothers, 17 and 11 years old, presented with pes cavus, absence of deep tendon reflexes, péripheral vibratory sensory loss, ataxia, tremor, nystagmus, dysarthria and partial myoclonic epilepsy. Electromyography showed severe slowing of motor conduction velocity in the lower extremities and increased distal latencies. A peroneal nerve biopsy showed absence of myelin sheath in most fibres resulting in numerous demyelinated nerve fibres. The father and seven uncles on the paternal side had pes cavus, hammer toes and moderate vibratory peripheral sensory loss. Three of seven siblings had slow motor conduction velocities on EMG. None had EEG abnormalities. Epilepsy started at an early age in both patients with myoclonic jerks of the right arm especially during sleep. EEG recordings were characterized by focal or diffuse epileptiform discharges. In the elder brother a partial motor epileptic status occurred with adversive seizures involving the right side of the body. He died of a broncopneumonia after 3 days of this epileptic status. Histopathological examination showed a severe demyelination of dentato-rubral pathways in the cerebellum and a partial degeneration of Goll and Burdach's tracts in the cervical spinal cord. The nosological classification of this syndrome is discussed and an autosomal dominant inheritance with incomplete penetrance or variable expressivity is suggested.

Myopathological findings in progressive myoclonus epilepsy.

8 patients with progressive myoclonic epilepsy have been studied. Muscle biopsies showed polysaccharide accumulation in branched form, mitochondrial alteration, lipid droplets and lipofucsin bodies. " Muscle Lafora bodies" were present in two cases. A peculiar muscle cytoskeleton appearance has been seen, suggesting an abnormal physico-chemical state of the newly observed filaments that joins Z-bands among them and with the plasma membrane.

Fatal lipid storage with abnormal mitochondria in an infant.

Biochemical, electron microscopic observations in muscle and liver of an infant dying at 20 months of age of a mitochondrial myopathy are described. Biochemical analysis of tissues revealed low levels of free, esterified and total carnitine and marked decrease of succinic-dehydrogenase activity while cytochrome c-oxidase was normal. Ultrastructural data showed a megaconial myopathy.

Morphogenesis of rat muscle spindles after nerve lesion during early postnatal development.

The influence of innervation on muscle spindle morphogenesis has been investigated in rat hind-limb muscles by sectioning the sciatic nerve, with suture of the stumps, at various postnatal stages. After nerve section at 4 or 7 days of age a proportion of spindles survived during the denervation phase and developed, during the subsequent reinnervation phase, into atypical structures. The reinnervated spindles were recognized by the presence of a limiting capsule but lacked the characteristic distinction of equatorial and polar regions. The intrafusal fibres were fewer than normal and were indistinguishable in size and fine structure from extrafusal fibres; they had a single motor endplate and lacked sensory nerve terminals. In reinnervated muscles of animals operated at 13 and 22 days of age there was a progressive tendency towards a restoration of normal spindle structure and innervation. These findings indicate that muscle spindle morphogenesis is profoundly altered by nerve lesion at early development stages, apparently as a result of inadequate sensory reinnervation. This study also shows that the differentiation of intrafusal fibres is dictated by their specific pattern of innervation and is not intrinsically predetermined.

[Types of fibers in skeletal muscles of mammals: histochemical and ultrastructural characteristics in relation to their contractile properties]. / Tipi di fibre nei muscoli scheletrici dei mammiferi: caratteristiche istochimiche e ultrastrutturali in rapporto alle proprieta' contrattili

A tentative correlation of histochemical and ultrastructural features with physiological properties in mammalian skeletal muscle fibers is presented. In particular, the characteristic metabolic and structural profile of fast-twitch red fibers is described and evidence is presented for the presence of such fibers in limb muscles and masticatory muscles of the rat. The existence of interspecies and even intraspecies variation in the histochemical correlates of the different fiber categories is emphasized and the significance of intermediate fiber types is discussed.