Farewell to Professor David Yaffe - A pillar of the myogenesis field.

It is with great sadness that we have learned about the passing of Professor David Yaffe (1929-2020, Israel). Yehi Zichro Baruch - May his memory be a blessing. David was a man of family, science and nature. A native of Israel, David grew up in the historic years that preceded the birth of the State of Israel. He was a member of the group that established Kibbutz Revivim in the Negev desert, and in 1948 participated in Israel's War of Independence. David and Ruth eventually joined Kibbutz Givat Brenner by Rehovot, permitting David to be both a kibbutz member and a life-long researcher at the Weizmann Institute of Science, where David received his PhD in 1959. David returned to the Institute after his postdoc at Stanford. Here, after several years of researching a number of tissues as models for studying the process of differentiation, David entered the myogenesis field and stayed with it to his last day. With his dedication to the field of myogenesis and his commitment to furthering the understanding of the People and the Land of Israel throughout the international scientific community, David organized the first ever myogenesis meeting that took place in Shoresh, Israel in 1975. This was followed by the 1980 myogenesis meeting at the same place and many more outstanding meetings, all of which brought together myogenesis, nature and scenery. Herein, through the preparation and publication of this current manuscript, we are meeting once again at a "David Yaffe myogenesis meeting". Some of us have been members of the Yaffe lab, some of us have known David as his national and international colleagues in the myology field. One of our contributors has also known (and communicates here) about David Yaffe's earlier years as a kibbutznick in the Negev. Our collective reflections are a tribute to Professor David Yaffe. We are fortunate that the European Journal of Translational Myology has provided us with tremendous input and a platform for holding this 2020 distance meeting "Farwell to Professor David Yaffe - A Pillar of the Myogenesis Field".

Regulation of phosphorylase kinase by low concentrations of Ca ions upon muscle contraction: the connection between metabolism and muscle contraction and the connection between muscle physiology and Ca-dependent signal transduction.

It had long been one of the crucial questions in muscle physiology how glycogenolysis is regulated in connection with muscle contraction, when we found the answer to this question in the last half of the 1960s. By that time, the two principal currents of muscle physiology, namely, the metabolic flow starting from glycogen and the mechanisms of muscle contraction, had already been clarified at the molecular level thanks to our senior researchers. Thus, the final question we had to answer was how to connect these two currents. We found that low concentrations of Ca ions (10(-7)-10(-4) M) released from the sarcoplasmic reticulum for the regulation of muscle contraction simultaneously reversibly activate phosphorylase kinase, the enzyme regulating glycogenolysis. Moreover, we found that adenosine 3',5'-monophosphate (cyclic AMP), which is already known to activate muscle phosphorylase kinase, is not effective in the absence of such concentrations of Ca ions. Thus, cyclic AMP is not effective by itself alone and only modifies the activation process in the presence of Ca ions (at that time, cyclic AMP-dependent protein kinase had not yet been identified). After a while, it turned out that our works have not only provided the solution to the above problem on muscle physiology, but have also been considered as the first report of Ca-dependent protein phosphorylation, which is one of the central problems in current cell biology. Phosphorylase kinase is the first protein kinase to phosphorylate a protein resulting in the change in the function of the phosphorylated protein, as shown by Krebs and Fischer. Our works further showed that this protein kinase is regulated in a Ca-dependent manner. Accordingly, our works introduced the concept of low concentrations of Ca ions, which were first identified as the regulatory substance of muscle contraction, to the vast field of Ca biology including signal transduction.

Our trails and trials in the subsarcolemmal cytoskeleton network and muscular dystrophy researches in the dystrophin era.

In 1987, about 150 years after the discovery of Duchenne muscular dystrophy (DMD), its responsible gene, the dystrophin gene, was cloned by Kunkel. This was a new substance. During these 20 odd years after the cloning, our understanding on dystrophin as a component of the subsarcolemmal cytoskeleton networks and on the pathomechanisms of and experimental therapeutics for DMD has been greatly enhanced. During this paradigm change, I was fortunately able to work as an active researcher on its frontiers for 12 years. After we discovered that dystrophin is located on the cell membrane in 1988, we studied the architecture of dystrophin and dystrophin-associated proteins (DAPs) complex in order to investigate the function of dystrophin and pathomechanism of DMD. During the conduct of these studies, we came to consider that the dystrophin-DAP complex serves to transmembranously connect the subsarcolemmal cytoskeleton networks and basal lamina to protect the lipid bilayer. It then became our working hypothesis that injury of the lipid bilayer upon muscle contraction is the cause of DMD. During this process, we predicted that subunits of the sarcoglycan (SG) complex are responsible for respective types of DMD-like muscular dystrophy with autosomal recessive inheritance. Our prediction was confirmed to be true by many researchers including ourselves. In this review, I will try to explain what we observed and how we considered concerning the architecture and function of the dystrophin-DAP complex, and the pathomechanisms of DMD and related muscular dystrophies.

Molecular and cell biology of the sarcoglycan complex.

The original sarcoglycan (SG) complex has four subunits and comprises a subcomplex of the dystrophin-dystrophin-associated protein complex. Each SG gene has been shown to be responsible for limb-girdle muscular dystrophy, called sarcoglycanopathy (SGP). In this review, we detail the characteristics of the SG subunits, and the mechanism of the formation of the SG complex and various molecules associated with this complex. We discuss the molecular mechanisms of SGP based on studies mostly using SGP animal models. In addition, we describe other SG molecules, epsilon- and zeta-SGs, with special reference to their expression and roles in vascular smooth muscle, which are currently in dispute. We further consider the maternally imprinted nature of the epsilon-SG gene. Finally, we stress that the SG complex cannot work by itself and works in a larger complex system, called the transverse fixation system, which forms an array of molecules responsible for various muscular dystrophies.

ZZ domain is essentially required for the physiological binding of dystrophin and utrophin to beta-dystroglycan.

An intracellular protein, dystrophin, plays an important role in keeping muscle fibers intact by binding at its N-terminal end to the subsarcolemmal cytoskeletal actin network and via its C-terminal end to the transmembraneous protein beta-dystroglycan. Duchenne muscular dystrophy is caused by the loss of dystrophin, which can result from the loss of this binding. The N-terminal part of the latter binding site of dystrophin has been well documented using overlay assay and X-ray diffraction assays. However, the binding site at the C-terminal region of dystrophin has not been examined in detail. In the present work, we report a detailed analysis of the C-terminal binding domain as follows. (1). The full binding activity corresponding to the effective binding in vivo is expressed by the dystrophin fragment spanning amino acids 3026-3345 containing the ZZ domain at the C-terminus. Determination of this binding range is important not only for understanding of the mechanism of dystrophy, but also useful for the design of truncated dystrophin constructs for gene therapy. (2). The ZZ domain binds to EF1 domain in the dystrophin fragment to reinforce the binding activity. (3). The cysteine 3340 in the ZZ domain is essential for the binding of dystrophin to beta-dystroglycan. A reported case of DMD due to missense mutation C3340Y may be caused by inability to fix dystrophin beneath the cell membrane. (4). The binding mode of utrophin is different from that of dystrophin. The difference is conspicuous concerning the cysteine residues present in the ZZ domain.

Pathological analysis of muscle hypertrophy and degeneration in muscular dystrophy in gamma-sarcoglycan-deficient mice.

While calf muscle hypertrophy is a striking diagnostic finding in sarcoglycanopathy, as it is in Duchenne and Becker muscular dystrophies, its pathogenetic mechanism remains unknown. gamma-Sarcoglycan, one of the subunits of the sarcoglycan complex, is the protein responsible for gamma-sarcoglycanopathy. To elucidate the pathogenetic mechanisms of muscle hypertrophy and degeneration in muscular dystrophy, we utilized a mutant mouse as a model animal. In this study, we generated gamma-sarcoglycan-deficient (gsg-/-) mice by gene targeting. The gsg-/- mice described here, similar to the gsg-/- mice reported previously (J Cell Biol 142 (1998) 1279), demonstrated skeletal and cardiac muscle degeneration. The limb, shoulder, and pelvic muscles of the gsg-/- mice exhibited progressive muscle hypertrophy and weakness with age, and the findings were similar to those seen in other mouse models for limb-girdle and Duchenne muscular dystrophy. We found that the number of muscle fibers increased with age, and most of the fibers in the hypertrophic muscle were centrally nucleated regenerating fibers. Therefore, muscle hypertrophy of the gsg-/- mice may result from an increase of the number of muscle fibers and probable fiber branching and may not be due to the pseudohypertrophy caused by fibrous and fat tissue replacement, as has been long supposed in muscular dystrophy. The muscle pathology became more 'dystrophic' in mice over 1 year of age when there was a marked variation in fiber size with interstitial fibrosis.

Muscular dystrophies.

PURPOSE OF REVIEW: Muscular dystrophy includes many genetically distinct disorders. The list of causative genes for muscular dystrophy has been expanding rapidly, including those for congenital muscular dystrophies. RECENT FINDINGS: We review the newly identified causative genes and suggested molecular mechanisms, focusing on glycosylation abnormality of alpha-dystroglycan, collagen VI deficiency, four allelic diseases of caveolin-3 gene, and titin gene mutations. SUMMARY: Several possible mechanisms causing muscular dystrophy were discussed. Defects in extracellular molecules have more significant effects resulting mainly in congenital muscular dystrophy, while intracellular molecular defects show milder effect on the phenotype. These hypotheses may provide a new paradigm in understanding the pathomechanism of muscular dystrophies.

A New Method for Fibroblast-less Primary Skeletal Muscle Cell Culture by the Use of Hydroxyurea: (skeletal muscle cell culture/hydroxyurea/muscle cell differentiation/myosin heavy chain/dystrophin).

A method was developed to suppress growth of fibroblasts in chicken and mouse primary skeletal muscle cell cultures. Addition of hydroxyurea to the culture medium at appropriate time and concentrations suppressed the proliferation of fibroblasts whereas leaving myotubes grow and differentiate. The most favorable time for the addition was soon after myotube formation. The optimal concentrations for our purpose ranged from 0.5 to 1.0 mM. In the presence of hydroxyurea at these concentrations, myotubes grew larger and well differentiated, whereas fibroblasts remained in the suppressed state. In chicken myotubes cultured with hydroxyurea, cross-striations, spontaneous twitching and myosin heavy chain appeared as in myotubes without hydroxyurea. In mouse myotubes cultured with hydroxyurea, myosin heavy chain and dystrophin appeared, as in control myotubes.

Vimentin Expression Pattern is Different between the Flank Region and Limb Regions of Somatopleural Mesoderm in the Chicken Embryo: (vimentin/chicken embryo/limb bud/limbness/mesoderm).

It is known that the chicken flank somatopleure also has a limb-forming potential at early stages of development, but loses this potential later. Molecular changes during this process is, however, not well known. We obtained a monoclonal antibody which reacts to the flank somatopleure, but not to the wing bud, the leg bud and the neck somatopleure in the stage 22 chicken embryo. Further study revealed that this antibody is specific to vimentin. Time course of vimentin expression in the somatopleural mesoderm during the development was studied. It was revealed to be biphasic. Somatopleural mesoderm expressed vimentin at stage 10, but not at stage 16. Flank somatopleural mesoderm began to express vimentin again at stage 18, whereas limb bud mesenchymal cells did not until stage 27. The earlier re-expression of vimentin at the flank somatopleure suggests that certain physiological changes take place in cells at this region.

Vital labelling of somite-derived myogenic cells in the chicken limb bud.

In order to understand how myogenic cells migrate in the limb bud, it is indispensable to distinguish undifferentiated myogenic cells from other mesenchymal cells. Thus, a suitable method for this purpose has been sought. A method to exchange the somites of a chicken and a quail microsurgically has widely been used, since the nuclei of the two species are morphologically distinguishable. However, microsurgery is accompanied by disturbances at the operated locus, and introducing cells of different species might induce unexpected effects. We report a new method for labelling chicken myogenic cells without transplantational operations, and describe their migration pattern in limb buds. Injection of a fluorescent carbocyanine dye into the somite lumen intensely labelled the somitic cells. Myogenic cells derived from the somite were clearly detected in limb buds. Before stage 20, the labelled cells were diffusely distributed in the proximal region of the limb bud. At about stage 21 in both wing and leg buds, labelled cells began to form dorsal and ventral masses. The label was followed until the cells differentiated and expressed myosin. This vital labelling method has advantages over the somite transplantation method: it does not include surgical operations that may disturb the normal development, and the cells are labelled intensely enough to be detected in a whole mount preparation.

Partial Purification from Chick Embryos of a Factor which Promotes Myoblast Proliferation and Delays Fusion: (myoblast proliferation/myoblast fusion/embryo extract/fibroblast growth factor).

Chick embryo extract (EE) contained an activity which promoted myoblast proliferation and delayed fusion. Various tissue extracts prepared from 12-day embryos and adult chicken also showed the activity. We partially purified this active substance from 12-day embryos, following procedures which included extraction at pH 3.5, CM-Sephadex C-50 ion exchange and Sephadex G-75 gel filtration. Judging from the dose-response analyses, the factor was purified by some hundred-fold when EE was used as the starting material. The activity was associated with a macro-molecular substance (MW > 300K daltons) at first, but the apparent molecular weight of the active substance was estimated to be between 16 and 20K daltons at the final step of the preparation. It promoted myoblast proliferation and delayed myotube formation, and was active for both avian and rat myoblasts. Since bovine pituitary gland fibroblast growth factor (FGF) showed the same activity, the factor may be FGF-related.

Promotion of Myoblast Proliferation by Hypoxanthine and RNA in Chick Embryo Extract: hypoxanthine/RNA/myoblast proliferation/embryo extract).

In the course of our attempt to clarify the growth-promoting activities of chick embryo extract (EE), its heat-stable activity was found to be due to hypoxanthine and its related substances including RNA. When added to a basal culture medium composed of Eagle's MEM, horse serum and Fe-saturated ovotransferrin hypoxanthine or adenine (10 µM) markedly promoted quail myoblast proliferation. The concentration of hypoxanthine in EE was very high (274±34µM) and increased 2-fold during incubation at 37°C, while that in horse serum was very low (<3 µM). Guanine, xanthine and pyrimidines were ineffective. The nucleosides and nucleotides of hypoxanthine and adenine were effective, but the deoxynucleosides strongly inhibited the proliferation of avian myoblasts. Further, RNA was also effective but DNA was not. Hypoxanthine and RNA also promoted rat myoblast proliferation and the deoxynucleosides did not inhibit rat myoblast proliferation. These findings suggest that a supply of raw materials for RNA synthesis is important for optimal proliferation of myoblasts.

Further Studies on the Developmental Change in Myotrophic Activity of Chicken Serum: Relation between Activity and Transferrin: (developmental change/myotrophic activity/chicken serum/transferrin).

As an extension of previous studies, we reexamined the developmental change in trophic activity of chicken serum on chicken myogenic cells in vitro and attempted to elucidate it on the basis of possible changes in serum transferrin (Tf), the myotrophic activity of which depends both on its concentration and on the level of its iron-saturation. The myotrophic activity was found to be low until the second week in ovo, then to increase rather abruptly to a plateau at about the time of hatching, and then to decrease to the adult level. Determination of the concentration and level of iron-saturation of serum Tf suggested that the change in myotrophic activity was mainly caused by these two parameters, though another factor(s) may also be involved.

Indispensability of Iron-bound Chick Transferrin for Chick Myogenesis in Vitro: (myogenesis/transfrrin/iron).

Myotrophic activity of highly purified chick transferrins (Tfs) to chick primary myogenic cells has been studied in a culture medium containing horse serum. Iron-binding to Tfs is indispensable for the activity. The removal of iron from Tfs gives rise to a complete loss of the activity and it is restored by iron-rebinding depending on the amount of bound iron. This result, combined with other physicochemical and immunological data, strongly, confirms that the myotrophic activity is exerted by the Tfs themselves, not by a contaminating material(s). It has been found that culture medium containing horse Tf which seems inadequate for the study of the biological effects of Tfs is, however, suitable for studies on chick Tfs, since horse Tf is inactive in promoting chick myogenesis. Terminal sialic acid residues are unrelated to myotrophic activity since Tfs with different numbers of residues (0, 1, and 2 moles/Tf molecule) are comprable in their activities. The mechanism of Tf action on cells and contradictions among previous papers as to the requirement of Tf for cell growth have been discussed from the viewpoint of an iron-donor with class-specificity.

Transferrin Receptor on Chick Fibroblast Cell Surface and the Binding Affinity in Relevance to the Growth Promoting Activity of Transferrin: (transferrin/receptor/cultured fibroblast/molecular recognition/class dependent specificity).

We examined the transferrin (Tf) receptor of chick skin fibroblasts using chick 125 I-Tf. When the cells were incubated with 125 I-Tf on ice, most of the cell-associated 125 I-Tf was found on the cell surface; on the other hand, a large part of it was located inside the cells when incubated at 37°C. By equilibrium binding assay, the number of Tf receptors per cell was determined as 6.7 × 103 . Dissociation constant was estimated to be 2.6 × 10-8 M. The binding of 125 I-Tf was competitively inhibited by the addition of chick unlabeled Tf. Weaker inhibition was observed when bovine Tf was used as a competitor. Horse Tf had no effect on the binding of chick Tf. This agrees well qualitatively with chick cell growth-promoting activites of these Tfs. Removal of Fe from Tf affected the affinity for its receptors. About 5- to 10-fold higher concentrations of chick apo-Tf was needed to achieve the same degree of inhibition of 125 I-Tf binding as that made by chick Fe-Tf.

Class Specificity of Avian and Mammalian Sera in Regards to Myogenic Cell Growth in vitro: POSSIBLE ROLE OF TRANSFERRIN IN THE SPECIFICITY (class specificity of serum/cell growth/culture medium/muscle cell culture).

Chick myogenic cells grew in the presence of a small amount of avian serum in a culture medium composed of Eagle's minimum essential medium (MEM) and horse serum. Mammalian sera, except for fetal bovine serum at high concentrations, could not substitute for the avian serum. Rat myogenic cells grew in the presence of a small amount of mammalian serum in a culture medium composed of MEM and chick serum: avian sera, except for dove serum at high concentrations, could not substitute for the mammalian serum. Serum from animals of the class from which the myoblasts were obtained was needed for cell growth. It is thus concluded that there is a class specificity among sera in regards to myogenic cell growth. The only exceptions to this hypothesis found so far were fetal bovine and dove sera.

Indispensability of Iron for the Growth of Cultured Chick Cells: (iron/transition metal/transferrin/chick embryonic cell/myogenesis).

In order to clarify the role of iron in the growth promoting effect of transferrin (Tf), the effects of the following substances were examined in cultured chick skeletal myogenic cells: transition metal ions (Fe2+ , Fe3+ , Cr3+ , Cu2+ , Mn2+ , Co2+ , Cd2+ , Zn2+ and Ni2+ ), Tf complexes with these metals and metal-free apoTf. The cells did not grow well when incubated in a culture medium composed of Eagle's minimum essential medium and horse serum. But they grew well in the presence of Fe2+ or Fe3+ (10-100 µM) or iron-bound Tf (10-500 nM) in the medium. None of the transition metal ions other than iron was effective. Neither apoTf nor Tf complexes with these metals showed the growth promoting effect. The generality of the requirement of iron for cell growth was ascertained in the primary culture of other types of chick embryonic cells: fibroblasts, cardiac myocytes, retinal pigment cells and spinal nerve cells. The results show that iron is one of the indispensable substances for cell growth and suggest that Tf protein plays a role in facilitating the transport of iron into the cells.

Chick Embryo Extract, Muscle Trophic Factor and Chick and Horse Sera as Environments for Chick Myogenic Cell Growth.

Chick myogenic cells grew in a medium composed of Eagle's minimum essential medium (MEM), horse serum (HS), and one of the essential factors needed for myogenic cell growth (EFMG), that is, chick embryo extract (EE), chick serum (CS), or the muscle trophic factor (MTF). But they did not grow in the absence of the EFMG. In the absence of HS, they scarcely grew in a medium composed of MEM, and EE or MTF. They grew in a medium composed of MEM and CS; they grew much better in a medium composed of MEM, CS, and HS. In the presence of one of the EFMG, the optimal HS concentration for growth varied depending on its lot. At higher HS concentrations, growth was suppressed. Further, it was suggested that an inhibitory substance(s) for myogenic cell growth was present in HS. The inhibitory effects can usually be minimized by diluting the serum with an artificial medium.

DIFFERENCES IN SENSITIVITY TO CA ION LACK BETWEEN MYOBLASTS AND LARGE MYOTUBES FROM CHICKEN BREAST MUSCLE.

Large myotubes degenerated in Ca-deficient medium containing Mg ion. Numerous vacuoles appeared in the cytoplasm and then grew larger. The cells were disrupted and eventually detached from the culture dish. The time course of the destruction process differed from cell to cell and the rate of reduction of creatine kinase in the culture dish was constant irrespective of the time after the removal of Ca ion. Most of the myoblasts survived in Ca-deficient medium, after almost all the large myotubes had disappeared. These myoblasts fused to form new myotubes when Ca ion was supplied. Myotubes formed from myoblasts which had been cultured with Ca-deficient medium for 2 weeks also degenerated on Ca removal. Sr ion added to Ca-deficient medium did not completely prevent the destruction of myotubes but decreased the rate of their reduction.

A STATISTICAL METHOD TO COMPARE THE DEGREE OF MUSCLE CELL MULTIPLICATION IN DIFFERENT CULTURE DISHES.

A method was developed for comparing two groups of numbers of cultured muscle cells which were counted under a microscope. Practically important problems for this purpose were: how many fields per dish should be observed, and how many dishes should be prepared under the same conditions, when given test criteria were set. In the present experiment, 4 dishes were prepared under the same conditions. From each of the dishes, 20 fields were selected, and the numbers of muscle cells*** were counted and separately recorded. Since the purpose was to compare two groups of dishes, the design was a simple case of a nested one. From the experiment, the type of distribution seemed approximately a long-normal distribution with constancy of variance (homoscedasticity). Since the distribution of the cells in dishes belonging to the same group could be considered to be the same, the numbers from each dish could be pooled within a group. Therefore, if the test criteria for Student's t test and the sensitivity to descriminate the ratio of the number of groups are given, the number of fields to be observed per dish times that of dishes can be uniquely determined. This method can be applied for the same purpose to other kinds of cells with log-normal distribution.