Unusual familial cardiomyopathy with storage of intermediate filaments in the cardiac muscular cells

Summary Unusual histological and ultrastructural changes in cardiac muscle cells have been found in 3 brothers with progressive myocardial deficiency. Histologically, this cardiomyopathy was characterized by massive storage of PAS-negative proteinaceous material in most cardiac muscle cells. The electron microscope showed that this material consisted of sinuous filaments, 7–10 nm in diameter, similar to the intermediate filaments normally present in cardiac muscle cells. Filament storage coincided with the disintegration of neighbouring myofibrils, with particular change in Z bands giving rise to rod-like bodies and more complex structures formed by the association of Z band material and sarcoplasmic reticulum (SR) tubules. Filament storage and myofibrillar disintegration always occurred in areas where the SR developed and involuted extensively. Relatively high glycogen accumulation also occurred, in close relation to the SR changes. Discrete SR proliferation, glycogen overload and filament deposits were observed in a few skeletal fibres.These observations suggest that disturbance in the metabolism of desmin (protein subunit of intermediate filaments and a fundamental component of Z bands) might be involved in this type of cardiomyopathy. The influence of a chronic defect in calcium regulation might also be envisaged in view of the marked SR abnormalities.     

Heart development in the mexican salamander,Ambystoma mexicanum. II. Ultrastructure

Heart development in the Mexican axolotl, Ambystoma mexicanum, from early embryonic stages to the adult was studied by electron microscopy. During early development, myocardial cells exhibit scanty cytoplasmic matrixes which contain numerous large yolk platelets and lipid droplets. As development progresses the yolk and lipid materials become reduced; the cytoplasmic matrixes increase in glycogen content and membranous organelles. Degradation of yolk platelets appears to take place by an “unraveling” process. The Golgi complex becomes well-developed just prior to the first heart beats and its many associated vesicles are suggestive of secretory activity. Degeneration and death of certain myocardial cells are evident at the onset of trabeculation. Several mechanisms for myofibrillogenesis appear to operate both simultaneously and at different developmental stages. Pre-heart-beat myocardial cells display ribosome-containing amorphous masses and unorganized 60 Å and 140 Å filaments in their cytoplasm. They also have electron-opaque plaques on their plasma membranes. Sarcomeric myofibrils first appear parallel to and immediately beneath the sarcolemma. This occurs at stage 34, the heart-beat initiation stage (6 days). During later development “isolated” Z band-myofilament complexes, numerous loosely-organized 60 Å filaments and a few 140 Å filaments are observable in the cell matrixes. This suggests that Z bands may form centers for myofibril organization at these stages. Also at later stages, polysomes are parallel to the already-formed myofibrils. These polysomes are possibly synthesizing contractile proteins “in situ” and may represent a mechanism for myofibril diameter growth. In late embryonic and juvenile stages 100 Å—110 Å filaments appear to be continuous between Z bands of adjacent myofibrils. It is possible that such filaments of intermediate size are involved in aligning myofibrils into register during advanced developmental stages.     

Incorporation of microinjected biotin-labelled actin into nascent myofibrils of cardiac myocytes: an immunoelectron microscopic study

Summary Incorporation of microinjected biotin-labelled actin into nascent myofibrils of cultured cardiac muscle cells was investigated by immunogold electron microscopy. At the proximal parts of myofibrils, gold labelling was first found (at about 4 min after injection) around the A-band level. This observation suggests that polymerization of actin or the addition of newly-formed actin filaments occurs preferentially in association with myosin filaments to increase the myofibrillar girth. The distal terminals of developing myofibrils were also labelled at about 4 min after injection. This rapid incorporation of actin subunits at the myofibrillar ends suggests the continued reorganization and/or de novo formation of myofibrils at these positions. Along the extending direction of the myofibrillar terminals, gold particles were arranged in rows on the inner surface of the sarcolemma. These rows of particles continued to become longer with incubation. It appears that actin subunits are added at the membrane-associated ends of pre-existing actin filaments to increase the length of myofibrils.     

How to build a myofibril

Building a myofibril from its component proteins requires the interactions of many different proteins in a process whose details are not understood. Several models have been proposed to provide a framework for understanding the increasing data on new myofibrillar proteins and their localizations during muscle development. In this article we discuss four current models that seek to explain how the assembly occurs in vertebrate cross-striated muscles. The models hypothesize: (a) stress fiber-like structures as templates for the assembly of myofibrils, (b) assembly in which the actin filaments and Z-bands form subunits independently from A-band subunits, with the two subsequently joined together to form a myofibril, (c) premyofibrils as precursors of myofibrils, or (d) assembly occurring without any intermediary structures. The premyofibril model, proposed by the authors, is discussed in more detail as it could explain myofibrillogenesis under a variety of different conditions: in ovo, in explants, and in tissue culture studies on cardiac and skeletal muscles.In memoriam: This paper is dedicated to the memory of Professor Koscak Maruyama, a noted contributor in the field of muscle biochemistry.     

Does phosphate release limit the ATPases of soleus myofibrils? Evidence that (A)M· ADP·Pi states predominate on the cross-bridge cycle

The ATPases (±Ca²⁺) of myofibrils from rabbit soleus (a slow muscle) and psoas (a fast muscle) have different E _a: −Ca²⁺, 78 and 60 kJ/mol and +Ca²⁺, 155 and 71 kJ/mol, respectively. At physiological temperatures, the two types of myofibrillar ATPase are very similar and yet the mechanical properties of the muscles are different (Candau et al. (2003) Biophys J 85: 3132–3141). Muscle contraction relies on specific interactions of the different chemical states on the myosin head ATPase pathway with the thin filament. An explanation for the E _a data is that different states populate the pathways of the two types of myofibril because the rate limiting steps are different. Here, we put this to the test by a comparison of the transient kinetics of the initial steps of the ATPases of the two types of myofibril at 4°C. We used two methods: rapid flow quench (`cold ATP chase': titration of active sites, ATP binding kinetics, k <sub>cat</sub>; `P_i burst': ATP cleavage kinetics) and fluorescence stopped-flow (MDCC-phosphate binding protein for free P_i; myofibrillar tryptophan fluorescence for myosin head-thin filament detachment and ATP cleavage kinetics). We find that, as with psoas myofibrils, the most populated state on the cross-bridge cycle of soleus myofibrils, whether relaxed or activated, is (A)M·ADP·P_i. We propose a reaction pathway that includes several (A)M·ADP·P_i sub-states that are either `weak' or `strong', depending on the mechanical condition. This revised version was published online in July 2006 with corrections to the Cover Date.     

Thick myofilament mass determination by electron scattering measurements with the scanning transmission electron microscope

Summary An accurate value for mass/length of thick myofilaments is required to establish a limit for the maximum number of myosin molecules per crossbridge repeat. The mass/length of the crossbridge regions of desalted thick myofilaments from insect flight muscle (Lethocerus andMusca) and rabbit psoas has been measured with a computer-linked STEM by comparing the electron scattering signal per unit length of unstained thick filaments with that from TMV particles in the same image. Filament preparation was aided by limited digestion of myofibrils to remove Z bands using calcium-activated factor (CAF) from rabbit skeletal muscle; SDS gels showed that this selective protease spared myosin and tended to spare paramyosin but removed C protein.Lethocerus filaments prepared by the CAF procedure were 20–25% heavier per unit length than those prepared by conventional (simple) shearing, and retained a clear and generally uniform 14.5 nm crossbridge repeat by negative staining.We have expressed mass/length of thick filaments as myosin equivalents (mol. wt 0.470 × 10⁶) per crown (that is, the 14.5 nm insect or 14.3 nm vertebrate repeat along thick filaments). TMV standards, calculated to weigh 0.1304×10⁶ daltons nm^–1 and thus equivalent to 4.02 myosins per 14.5 nm, were uniform to ±3%s.d. for 73 particles after normalizing means for each different image field. After subtracting the known paramyosin content from insect measurements (11% for waterbug, 2% for the housefly), but making no C protein correction to rabbit measurements, the following results were obtained:Lethocerus (all) 4.19±0.50 (243 filaments);Lethocerus (CAF prepns) 4.40±0.44 (145 filaments);Musca (all CAF) 4.14±0.37 (57 filaments); rabbit (all CAF) 2.86±0.34 (75 filaments).These values favour the lowest integral number of myosins per crown among currently competing models of thick filament structure. The rabbit value agrees with several previous estimates, including the STEM measurements of Lamvik, which indicated three rather than four myosins/crown. The insect flight myofilament value of four forces re-evaluation of previous estimates by quantitative gels and quantitative microscopy of whole fibrils which had favoured six myosins/crown.     

Cytological differentiation of human fetal skeletal muscle

The ultrastructural differentiation of several different muscles was investigated in human fetuses ranging in age from 13 weeks to neonatal. At approximately 16 weeks of gestation cell clusters containing both myotubes and satellite cells lie enclosed by a newly formed basal lamina and show evidence of fusion. The development of organelles is evident in myoblasts, proceeds as the cells transform into myofibers, and continues in the neonate. Filament synthesis occurs primarily in the cell periphery where thin filaments appear to align themselves in relations to parallel arrays of ribosome-studded thick filaments; Z line formation follows the appearance of thin filaments. Intermediate filaments, approximately 10-12 nm thick, were also consistently observed in perinuclear regions and distal to filament assembly. Although sarcoplasmic reticulum (SR) development is closely related to fibril formation, connections between Z lines and SR are not consistent, thus supporting the conclusion that SR does not evoke the formation of the Z line. Bristlecoated vesicles appear to be the precursors of elements of the SR, possibly the lateral sacs. Development of the transverse tubules, as invaginations of the sarcolemma, is closely associated with the formation of lateral sacs since the latter occur along the sarcolemma as soon as transverse tubules appear. Cytological differentiation is similar, though not identical, in several different muscles. During the last trimester muscle fibers show some evidence of diversity, mainly of variation in Z line width. In general the results suggest that the sequence and stages of human myogenesis are similar to those of other species.     

Probing the coupling of Ca2+ and rigor activation of rabbit psoas myofibrillar ATPase with ethylene glycol

We have exploited solvent perturbation to probe the coupling of Ca²⁺ and rigor activation of the ATPase of myofibrils from rabbit psoas. Three techniques were used: overall myofibrillar ATPases by the rapid-flow quench method; kinetics of the interaction of ATP with myofibrils by fluorescence stopped-flow; and myofibrillar shortening by optical microscopy. Because of its extensive use with muscle systems, ranging from myosin subfragment-1 to muscle fibres, we chose 40% ethylene glycol as the relaxing agent. At 4°C, the glycol had little effect on the myofibrillar ATPase at low [Ca²⁺], but at high [Ca²⁺] the activity was reduced 50-fold, close to the level found under relaxing conditions, and there was no shortening. However, the ATPase of chemically cross-linked myofibrils (permanently activated even without Ca²⁺) was reduced only 3–4-fold. The lesser reduction of the ATPase of permanently activated myofibrils was also observed in single turnover experiments in which activation occurs by a few heads in the rigor state activating the remaining heads. The addition of ADP, which also promotes strong head-thin filament interactions, also activated the ATPase but only in the presence of Ca²⁺. Further experiments revealed that in 40% ethylene glycol, Ca²⁺ does initiate shortening but only with the aid of strong interactions and at temperatures above 15°C. This confirms that in the organized and intact myofibril, Ca²⁺ and rigor activation are coupled, as proposed previously for regulated actomyosin subfragment-1. This revised version was published online in September 2006 with corrections to the Cover Date.     

Characterization of ultrastructural and contractile activation properties of crustacean (Cherax destructor) muscle fibres during claw regeneration and moulting

Summary Long-(SL>6m) and short-sarcomere (SL<4m) fibres were isolated from the claw muscle of the yabby (Cherax destructor) during limb regeneration and at different stages of the moult cycle. Long-sarcomere fibres were more susceptible to the changes resulting from the moult-induced atrophy compared with the short-sarcomere fibres. Signs of atrophy included fibre erosion, loss of myosin filaments, a reduction in the diameter of myosin filaments and changes associated with the Z line. The intracellular structure of the fibres, however, remained intact in both fibre types. Fibres taken immediately prior to ecdysis could not be fully activated with Ca²⁺ or Sr²⁺ without breaking. In contrast fibres taken within 4 h after ecdysis could develop and maintain full force when activated by Ca²⁺ or Sr²⁺. The results suggest that loss of myofibrillar proteins via the moult-induced atrophy and/or events associated with fibre elongation may occur in the period just prior to ecdysis and that these changes may be responsible for the fibres inability to function during the premoult stage. Results from this study showed that short-sarcomere fibres add sarcomeres by at least two different mechanisms (1) transverse sarcomere splitting and (2) Z line splitting. Long-sarcomere fibres appear to be elongated by mechanism (s) other than those used by short-sarcomere fibres which possibly involve large electron dense structures which are positioned between the myofibrils and within the A and I bands. Results from the regenerating chelae limb bud showed that sarcomeres form from separate units comprising myosin filaments and actin filaments anchored into Z lines respectively. These sub-sarcomeric units then join together to form sarcomeres. Myofibril formation is aided by electron dense regions which are closely associated with the membrane system. These fibres although short in length and still within the non-functional limb bud could be activated by Ca²⁺ and Sr²⁺ suggesting that full fibre function exists before the chelae become functional. Regenerating muscle fibres consisted predominately of fibres with short-sarcomeres.     

Altered distribution of desmin filaments in hypertrophic cardiomyopathy: an immunohistochemical study.

Hypertrophic cardiomyopathy (HCM) is characterized by myofiber hypertrophy and disarray. Intermediate filaments play an important role in maintaining of normal cell shape. Desmin filaments have been detected in adult cardiomyocytes, and vimentin and keratin filaments in cardiomyocytes during embryonic development. The pattern of arrangement of intermediate filaments in HCM has not been reported. We examined the distribution of intermediate filaments in formalin-fixed tissue sections of the disarrayed myofibers from 10 hearts with HCM using an immunohistochemical technique and antibodies to desmin, vimentin, and high and low molecular weight keratins. The controls consisted of subaortic tissue from surgically explanted hearts of patients with ischemic heart disease. In the ischemic hearts, desmin was detected in the Z bands and intercalated disks. In all HCM cases, three patterns of staining for desmin were noted: (a) individual myocytes showing a parallel arrangement along Z bands; (b) focal myofibers with decreased or complete loss of labeling of Z bands; and (c) individual myocytes with intense granular cytoplasmic staining especially in disarrayed myofibers. No staining for vimentin or keratins was noted in the cardiomyocytes from either the HCM or ischemic cases. The altered arrangement of desmin filaments in the disarrayed cardiac muscle fibers may play a role in the altered contractility that occurs in patients with HCM.     

Genetic dissection of Drosophila myofibril formation: effects of actin and myosin heavy chain null alleles

We used null mutations of Drosophila actin and myosin genes to investigate two aspects of myofibril assembly. First, we eliminated all actin or myosin in flight muscles to evaluate contributions of thick and thin filaments to sarcomere formation. Results demonstrate that thick and thin filament arrays can assemble independently but that both are essential for sarcomeric order and periodicity. Second, we examined how filament stoichiometry affects myofibril assembly. We find that heterozygotes for actin (Act88F) or myosin heavy chain (Mhc36B) null alleles have complex myofibrillar defects, whereas Mhc36B-/+; Act88F-/+ double heterozygotes have nearly normal myofibrils. These results imply that most defects observed in single heterozygotes are due to filament imbalances, not deficits, and suggest that thick and thin filament interactions regulate myofibrillar growth and alignment.     

Structural and functional changes in myocardial thin filaments in experimental hypothyrosis

Fluorescence resonance energy transfer study revealed decreased intermonomer mobility of Ca-actin and Mg-actin filaments of myocardial myofibrils in myocardial dystrophy caused by diffuse endocrine disorders, e. g. hypothyrosis. Cis374 axial distance in Ca-actin filament protomer increased in hypothyrosis. Intracellular pH has no effect on intermonomer mobility of Ca-actin filament. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 5, pp. 522–524, May, 2007     

Paracrystals of myosin rod

Summary To help understand the packing of myosin tails in the backbone of the vertebrate striated muscle thick filament, paracrystals of myosin rod, a proteolytic fragment corresponding to the whole myosin tail, have been examined by electron microscopy and image analysis. Two types of paracrystal were observed. Type I paracrystals were similar to those seen by Mooset al. (1975;J. molec. Biol. 97, 19). These showed a 14-nm axial repeat, but yielded no other structural information. Type II paracrystals were long, flexible ribbons with more regularity. When negatively stained they exhibited a weak 43-nm axial striation and appeared to be composed of a layer of narrow filaments. Optical diffraction showed that the paracrystals had a rectangular unit cell of dimensions 43 nm axially and 12.4 nm laterally. Transverse sections indicated a paracrystal depth similar to the lateral dimension of the unit cell. Each unit cell contained two filaments arranged antiparallel and related by a two-fold screw axis perpendicular to the length, and in the plane of the ribbon. The filaments probably consist of parallel rod molecules related by axial displacements of 43 nm and higher multiples of 43 nm.The nature of these paracrystals indicates that the myosin tail alone can form structures like thick filament subfilaments. Their structure, based on distinguishable parallel and antiparallel rod interactions, was sensitive to pH and divalent cations in a similar way to the ionic effects on the structure of thick filaments. This behaviour suggests that some of the interactions present in the paracrystal are the same as those in the thick filament.     

Structural studies of the waves in striated muscle fibres shortened passively below their slack length

Summary Isolated skeletal muscle fibres ofRana pipiens were shortened below their slack length by longitudinal compression in a gelatine block, and examined by light and electron microscopy. Waves appeared sharply when the striation spacing (S) reached a critical value (about 2 µm) and increased in height with further compression down toS = 1.6 µm while the resting band pattern was maintained. The waves were plane, helical or irregular, with wave lengths of 5–15 striations. The Z lines usually ran perpendicular to the direction of the myofibrils to form wedge-shaped sarcomeres. The bending occurred mainly in the I band. The thin filaments ran stiffly for about 30 nm from the Z line and then bent toward the A band. The thick filaments bent very slightly, particularly at their tips. The edges of the A band were indistinct, and there were no dense lines at the A–I junction.The appearance of the individual sarcomeres resembled those in relaxed myofibrils at slack length, with no C_m bands. The H zone was only seen occasionally in the slack and wavy fibres examined. In very thin sections the individual thin filaments were seen to end in the pseudo-H zone, and not to cross the M line. There was a single array of not more than six thin filaments round each thick one in transverse sections of the M-line region.These observations suggest that the narrowing of the bands observed in fresh wavy fibres is due mainly to the obliquity of the myofibrils, and that the sarcomere length measured parallel to their axis is longer than the striation spacing. The relationship between sarcomere length and the length of the thin-filament complex is discussed.     

Principal forms of acute damage to cardiomyocytes according to polarization microscopy data

The principal forms of acute cardiomyocyte damage of metabolic and ischemic genesis are accompanied by changes in the contractile apparatus, which is reflected in double refraction of myofibrils. Thus, polarization microscopy is the most sensitive method allowing one to detect the early stages of cardiomyocyte damage. Individual types of cardiomyocyte lesions are identified on the basis of parallel histological studies and polarization and electron microscopy of prenecrotic alterations and myocardial infarction using experimental and autopsy material. They are: I, II, and III degree myofibrillar contracture, intracellular myocytolysis, primary lumpy degradation of myofibrils, and cytolysis. These types of damage form the morphological basis of acute myocardial pathology with a possible outcome of coagulative or colliquative necrosis. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, N ^o 1, pp. 4–13, January, 1996     

Ultrastructural differences between the working myocardial cells and the conduction system in the monkey heart

Ultrastructural differences between the working myocardial cells and Purkinje cells in the monkey have been studied by transmission and scanning electron microscopy. The working myocardial cells possess rich myofibrils and well-developed intercalated discs. By contrast, Purkinje cells, which are characterized by some myofibrils, rich glycogen and abundant intermediate filaments, are ensheathed by dense reticulin fiber sheaths. It is likely that the intermediate filaments and reticulin fiber sheaths act as a mechanical support to protect the Purkinje cells from external forces.     

Desmin myopathy, a skeletal myopathy with cardiomyopathy caused by mutations in the desmin gene

BACKGROUND: Myofibrillar myopathies, often referred to as desmin-related myopathies, are a heterogeneous group of inherited or sporadic distal-onset skeletal myopathies associated with cardiomyopathy. Among the myofibrillar proteins that characteristically accumulate within the muscle fibers of affected patients, the one found most consistently is desmin, a muscle-specific intermediate-filament protein responsible for the structural integrity of the myofibrils. Skeletal and cardiac myopathy develops in mice that lack desmin, suggesting that mutations in the desmin gene may be pathogenic. METHODS: We examined 22 patients from 8 families with dominantly inherited myofibrillar or desmin-related myopathy and 2 patients with sporadic disease and analyzed the desmin gene for mutations, using complementary DNA (cDNA) amplified from muscle-biopsy specimens and genomic DNA extracted from blood lymphocytes. Restriction-enzyme analysis was used to confirm the mutations. Expression vectors containing normal or mutant desmin cDNA were introduced into cultured cells to determine whether the mutant desmin formed intermediate filaments. RESULTS: Six missense mutations in the coding region of the desmin gene that cause the substitution of an amino acid were identified in 11 patients (10 members of 4 families and 1 patient with sporadic disease); a splicing defect that resulted in the deletion of exon 3 was identified in the other patient with sporadic disease. Mutations were clustered in the carboxy-terminal part of the rod domain, which is critical for filament assembly. In transfected cells, the mutant desmin was unable to form a filamentous network. Seven of the 12 patients with mutations in the desmin gene had cardiomyopathy. CONCLUSIONS: Mutations in the desmin gene affecting intermediate filaments cause a distinct myopathy that is often associated with cardiomyopathy and is termed "desmin myopathy." The mutant desmin interferes with the normal assembly of intermediate filaments, resulting in fragility of the myofibrils and severe dysfunction of skeletal and cardiac muscles.     

Rearrangement of intermediate filament network of BHK-21 cells infected with vaccinia virus

Summary Association between vaccinia virus (VV) structures and intermediate filaments in specific areas of the cytoplasm of infected cells (virus factories) suggests that VV infection interferes with the cellular architecture by modifying the intermediate filament network. To analyse this question, we examined the array of intermediate filaments of BHK-21 cells infected with VV by laser scanning confocal microscopy using an anti-vimentin mouse monoclonal antibody. We observed a marked reorganization of intermediate filaments around the nucleus of infected cells. Bidimensional analysis of³²PO₄-labeled intermediate filament proteins revealed that the acidic isoform of vimentin and two isoforms of desmin have increased phosphorylation levels in infected cells. Our results suggest that the reorganization of intermediate filaments observed during VV infection could be promoted by an increase in the phosphorylation level of the intermediate filament proteins, vimentin and desmin.     

Accumulated strain mechanism for length determination of thick filaments in skeletal muscle. I. Experimental bases

Summary The kinetics of dissociation of myosin from both ends of thick filaments in glycerinated skeletal muscle fibres and myofibrils was studied in the presence of MgATP by use of an optical diffraction method and phase-contrast microscopy. The dissociation velocity,v (=-dL/dt where L is the length of thick filaments at timet), increased with increasing KCl concentration (0.225 to 0.5 m), or increasing pH (6.5 to 8.0) but hardly changed with temperature (5 and 25° C), micromolar concentrations of Ca²⁺ or sarcomere length (2.4 and 2.75 m). Over a wide range of filament length, the dissociation velocity could be expressed byv ₀exp(L), wherev ₀ and are positive constants depending upon the dissociation condition. When the effects of crossbridge formation are minimized it was thus shown that the structural stability of thick filaments in a muscle fibre and a myofibril gradually decreases from the central part to the tips of the filaments. On the basis of these results we propose that the length of thick filaments is largely regulated by an accumulated strain mechanism in which the free energy of association of myosin molecules increases with filament length.