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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.34 n.2 Santiago  2001

http://dx.doi.org/10.4067/S0716-97602001000200018 

Muscle cell outside and inside: the nascent approach of a
clinician

RICARDO FADIC

Departamento de Neurología Facultad de Medicina P. Universidad Católica de Chile

Corresponding Author: Ricardo Fadic. Unidad Neuromuscular, Hospital Clinico de la Universidad Catolica de Chile. Marcoleta 367, Santiago, Chile. Phone: (56-2) 686-3155. FAX: (56-2) 632-3459. e-mail: rfadic@med.puc.cl

Received: May 20, 2001. Accepted: July 10, 2001

ABSTRACT

Understanding muscle cell in disease and health is an unfinished process. Following the lead of Jaime Alvarez, I have had the opportunity of working on two complementary approaches to this field. One is the study of muscle cell surface molecules. Both synaptic muscle molecules, such as the asymmetric form of acetylcholinesterase, and extrasynaptic molecules, such as the extracellular matrix proteoglycans, are regulated by the motor nerve activity. This illustrates one of Jaime's teachings: cell phenotypes are a dynamic process that reflects the influence of other cells (Alvarez, 2001). Proteoglycans have many functions, including growth factor receptors. Studying them in muscular dystrophy will contribute to the comprehension of the muscle regeneration failure, characteristic of this disease. Muscle cells are highly dependent upon energy production, and the mitochondriae produce most of it. These organelles are unique in having their own genome. Mutations in these genes have recently been recognized as the cause of human disease and originally in muscle pathology. The physiopathology of these diseases is summarized here.

Key terms: mitochondrial DNA; muscle disease; muscular dystrophy; proteoglycans

Ever since I left Jaime's laboratory, I have been battling my own windmills: trying to be creative in academic clinical medicine. To put it another way: to be a competent clinician who is also able to do original research. There is no attempt to deny Jaime's influence in these pursuits, even though he has always said that this is impossible. Taking after him, I plan to remain persistent. I trained as a neurologist, with a special interest in neuromuscular disorders, and I have worked mainly in two different areas: muscle cell surface and mitochondrial disorders. I shall review some aspects of both.

Mitochondrial disorders

The respiratory chain located in the inner mitochondrial membrane produces most of the cellular energy. Energy failure can potentially affect any organ system and this is reflected in the heterogeneity of mitochondrial disorders. The respiratory chain enzymes are genetically unique, as two genomes, the nuclear and the mitochondrial encode them. Heritable errors leading to mitochondrial dysfunction can arise from either one and both Mendelian and maternal inheritance are possible for these disorders. The 1988 of large deletions of mitochondrial DNA (mtDNA) in the Kearns-Sayre syndrome (Holt et al., 1988, Zeviani et al., 1988) and a missense mutation at nucleotide 11778 of mtDNA in Leber hereditary optic neuropathy (Wallace et al., 1988) initiated the understanding of the correlation between clinical syndromes and mtDNA molecular biology.

Mitochondrial DNA

The mtDNA is a closed, circular, double-stranded molecule of 16,569 nucleotides that contains only 37 genes. The guanosine-rich heavy strand codes for two ribosomal RNAs (12s and 16s), 14 transfer RNAs and 12 polypeptides. The cytosine-rich light chain strand codes for one polypeptide (ND6 subunit of complex I) and 8 transfer RNAs. Of the 13 polypeptides coded in mtDNA, 7 are subunits of Complex I, one subunit of Complex III, 3 subunits of Complex IV and 2 subunits of Complex V. All 4 subunits of Complex II (succinate-ubiquinone oxidoreductase) are encoded in the nuclear DNA. The mitochondrion has independent replication, transcription, and translation systems, which have both prokaryotic and eukaryotic features (Wallace, 1992). Mitochondrial DNA transcription starts from two promoters, one for each strand, located in the displacement loop. Both transcripts include the entire genome, and they are polycystronic, meaning that they contain multiple messenger RNAs (mRNAs) resembling the prokaryotic transcription. As the transfer RNAs (tRNAs) is read, they fold into their three-dimensional structures and are excised as free tRNAs, thereby also releasing the intervening mRNAs and rRNAs (Wallace, 1992). These mRNAs are then polyadenylated and translated on mitochondrial ribosomes. Like bacterial protein synthesis, mitochondrial protein synthesis is initiated with formylmethionine and is sensitive to the bacterial ribosome inhibitor chloramphenicol (Wallace, 1992). Mitochondrial DNA has a unique genetic code, with AUA read as methionine instead of isoleucine, UGA as tryptophan instead of "stop," and both AGA and AGG as "stop" instead of arginine (Anderson et al, 1981). Therefore mtDNA cannot be translated in the cytoplasmic compartment and nuclear DNA cannot be translated within the mitochondria.

Mitochondrial DNA genetics

Genetic diseases arising from mutations in the mtDNA are influenced in their manifestation by several distinctive features of mtDNA: maternal inheritance, polyplasmy, heteroplasmy, mitotic segregation and threshold effect.

Maternal inheritance

Mitochondrial DNA is almost exclusively maternally inherited in humans (Gillensten et al., 1991). The spermatozoon contains a few mitochondria, and there is evidence that they can be inherited at an insignificant level (Clayton et al., 1975). mtDNA does not recombine and has a poorly developed repair system (Michaels et al., 1982, Pettepher et al., 1991) and mutations accumulate sequentially along maternal lines. This fact has been used extensively in population genetics.

Polyplasmy

Every cell contains multiple mitochondria and each contains several mtDNAs. At mitosis mitochondria randomly distribute among daughter cells. Therefore population genetics represent a better approximation to mitochondrial genetics than Mendelian.

Heteroplasmy

In normal cells all mtDNA molecules are identical, a condition known as homoplasmy. When a mutation arises in mtDNA, an intracellular mixture of mutant and wild type molecules known as heteroplasmy is created. In general non-deleterious polymorphysms of mtDNA are homoplasmic. In contrast all deletions, and most of the pathogenic point mutations, especially in tRNA's, are heteroplasmic. Over many cell generations, heteroplasmic mtDNA genotypes drift toward either pure mutant or normal mtDNA populations (Holt et al., 1990, Lott et al., 1990).

Mitotic segregation

As heteroplasmic cells undergo mitotic or meiotic division, the proportion of mutant and normal mtDNA allocated to daughters cells shifts. This phenomenon can partially explain the different phenotypic expression of a mtDNA mutation between different tissues and during time. The opposite occurs in muscle, a postmitotic tissue, where it has been shown that the proportion of the deleted mtDNA increases in sequential muscle biopsies (Larsson et al., 1990). Mitotic segregation also explains the markedly different levels of mutated mtDNA in various members of the same family (Ciafaloni et al., 1992), and among different tissues within an individual (MacMillan et al., 1993).

Threshold effect

The onset and severity of clinical manifestations depend on a delicate balance between the energy supply (determined in part by the proportion of mutant and normal mtDNA) and oxidative demands of different organ systems. In vitro studies have supported that a certain level of mutant mtDNA must be surpassed before a cell expresses an oxidative phosphorylation defect (Attardi et al, 1995). This explains that organs more dependent on oxidative metabolism, such as the brain, retina, heart and skeletal muscle, are more frequently affected.

Mitochondrial encephalomyopathies: pathophysiology

The prevailing view of the pathogenesis of mitochondrial diseases is that there is a progressive decline in mitochondrial ATP-generating capacity, leading to energetic failure, and eventual cell death in affected tissues. King and Attardi (King and Attardi, 1988) created a human cell line depleted of mtDNA by exposure to ethidium bromide, which inhibits mtDNA replication. Using these cell lines it has been possible to produce mitochondria with known proportions of mutant and wild type mtDNA. These constructs show defects in mitochondrial protein synthesis, impairment of oxidative phosphorylation, or both (Chomyn et al., 1991, Chomyn et al., 1992, Hayashi et al., 1991). mtDNA is particularly susceptible to the mutagenic effects of free radicals due to its proximity to the respiratory chain, few non-coding sequences (Anderson et al, 1981) , lack of protective histones (Caron and Rouviere-Yaniv, 1979), and limited DNA repair mechanisms (Ames, 1989, Clayton et al., 1975, Pettepher et al., 1991). The accumulation of mtDNA mutations might result in decreased activity of the oxidative phosphorylation enzymatic complexes which contains mtDNA-encoded subunits. This would produce a self-perpetuating and amplifying cycle of respiratory chain dysfunction and free radical production. mtDNA has a mutation rate that is at least 10 times that of the nuclear genome (Wallace, 1992).

Aging in humans is associated with an increase in the number of cytochrome c oxidase-deficient fibers in skeletal muscle (Muller-Hocker, 1990) and a decline in both the respiratory rate and oxidative phosphorylation enzyme activities (Trounce et al., 1989). Evidence for cumulative mtDNA oxidative damage comes from the increased frequency of deletions, (Cortopassi and Arnheim, 1990, Simonetti et al., 1992) point mutations (Zhang et al., 1993), and oxidized forms of mtDNA (Hayakawa et al., 1991, Mecocci et al., 1993) with aging. mtDNA mutations are then likely involved, and there is good preliminary evidence supporting it, in neurodegenerative disorders.

In Donald Johns' laboratory at the Harvard Institutes of Medicine, we worked trying to expand the phenotypes known associated to mtDNA mutations. We gathered a group of unrelated patients with a severe sensory ataxic neuropathy associated with dysarthria and chronic progressive ophthalmoplegia. The multiplicity of systems affected suggested a mitochondrial disorder. Molecular genetic analysis showed multiple mtDNA deletions in muscle and nerve samples (Fadic et al., 1997). This type of mutations is most likely secondary to a defect in mtDNA replication. The proteins involved are nuclear encoded and have not yet been identified.

We found a mutation in the mtDNA nucleotide position 4269 in the tRNAIle in a patient with psychosis followed by a slowly progressive dementia and extrapyramidal signs. I participated in her care while I was at the University of Wisconsin-Madison. She was the second patient with that mutation. The first was a Japanese boy who suffered seizures and heart failure (submitted). We identified two new mutations in mtDNA tRNA genes in patients with isolated hypertrophic cardiomyopathy who did not have mutations in the contractile proteins system (in preparation).

The primary interest of the laboratory was the work in Leber's hereditary optic neuropathy (LHON). One aspect of the work I was involved in was to look for mutations in patients with LHON who did not have the known primary mtDNA mutations. My original research work back in Chile was along this line. We studied Chilean patients with unexplained optic neuropathy both the presence of mtDNA mutations related to LHON and their mtDNA haplotype. Most of the Chilean population belongs to an Amerindian mtDNA lineage, and we found that 9 of 44 (21%) patients with bilateral simultaneous optic neuropathy (BSON) had a mtDNA mutation. Eight had the mtDNA nucleotide position 11778 mutation, and one had the 14484 mutation. All of them belonged to the mtDNA Amerindian haplotype D. These results confirm the pathogenicity of the 11778 and 14484 mutations, extend it to the Amerindian mtDNA and suggest that they have originated independently in several mtDNA lineages. The predominance of haplotype D in these patients raises the possibility that it may promote the expression of optic neuropathy, as the Caucasian haplotype J does in that population (Luco et al., 2001). There is much work to do in the horizon in this line of research.

Muscle cell surface

Cells interact with other cells through their surfaces. The study of this segment of the cellular system has significantly increased the comprehension of both physiological and pathological cellular events. The trophic influence exerted by the nerve cell over the muscle is well known. Joaquin Luco, one of Jaime's primary influences,

contributed to the understanding of this physiological effect half a century ago (Luco and Eyzaguirre, 1955). The idea to evaluate the effect of the nerve function on muscle features has since lingered in the halls and laboratories of the Universidad Católica. In Nibaldo Inestrosa's laboratory, we showed that the two forms of the muscle synaptic acetylcholinesterase behave differently in response to denervation and reinnervation suggesting distinct locations and functions (Fadic and Inestrosa, 1989).

In the process of doing that work, we ran into an unexpected phenomenon.: the acetylcholinesterase level increased in muscles contralateral to denervated ones. This transynaptic effect is still not fully understood (Fadic et al., 1988). At that time Enrique Brandan returned from his postdoctoral stay abroad, and we collaborated on a study of the possible role of nerve function of molecules located in the muscle extra-cellular matrix. We found that motor nerve function decreases the synthesis of muscle proteoglycans (Fadic et al., 1990). Understanding normal function is the basis for comprehending pathology. Muscle fiber degeneration or dystrophy is one of the main pathological processes affecting muscle. The term muscular dystrophy includes a diverse group of inherited disorders characterized by a progressive muscle weakness in which the primary defect is thought to be in the muscle. The involvement of different muscle groups, natural history, mode of inheritance, and age of onset was used to classify the different forms of muscular dystrophy. This classification was unsatisfactory, as there is a wide intra-familial phenotypical heterogeneity and a marked overlap in clinical manifestations among the different types. Since the identification of dystrophin in 1988 by Kunkel (Koenig et al., 1988), and the dystrophin-glycoprotein complex the year later by Campbell and Kahl, 1989, the classification has shifted to molecular genetics. An increasing number of proteins in different subcellular locations, primarily related to muscle surface, have been identified as responsible for the various types of muscle dystrophies. For a recent review see Cohn and Campbell (2000). The task before us is to understand the cause of cell death and the failure of muscle cells to regenerate under these conditions. This necessarily will go through cell and molecular biology.

During my clinical training in the United States there was no real time for research until my neuromuscular fellowship year. One of the advantages for a clinician of having a training in basic sciences is to be more aware of current research paradigms. I was responsible for the care of a patient referred to the University of Wisconsin Hospital with a myopathy and terminal congestive heart failure. He was diagnosed with a Becker muscular dystrophy. Reviewing his muscle biopsy material, I found in a Western blot that he had a normal dystrophine, the subsarcolemmal protein that is mutated in this disease. The patient later had a heart transplant. In both heart and skeletal muscle we demonstrated that the protein responsible for his condition was alpha-sarcoglycan, known at the time as adhalin (Fadic et al., 1996). In these patients, as well as in those in whom the muscular dystrophy was secondary to the absence of merosin, we described by EM, sarcolemmal defects that we postulated are the final common pathway for the muscle necrosis (Fadic et al., 1997b).

Reinitiating the collaboration with Enrique Brandan, we have recently been working on the characterization of proteoglycan expression in human muscular dystrophy. He has made important contributions in this field in the mdx mouse, the animal model for muscular dystrophy. Proteoglycans are known to interact with trophic factors and we postulate they will be involved in the pathogenesis of the dystrophic process.

 

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