Biochemistry of neuromuscular diseases: A course for undergraduate students*

ARTICLE

Establishing the relationship between teaching and research is crucial in instructing science students. Even in introductory courses it is useful to emphasize the wider ramifications of the study of biological disciplines, including the enormous impact of biomedical research in lowering world-wide mortality and morbidity rates on both humans and animals. This includes analysis of the basic principles of biological research strategies and of how modern approaches integrate biological knowledge from the molecular, tissue, systems, and disease level. As our laboratory is involved in the elucidation of the molecular mechanisms underlying muscular diseases, we have developed a new undergraduate course on the biochemistry of neuromuscular diseases. This unit has been taught for 7 years as a two-course series to 3rd- and 4th-year students of a 4-year Honors B.Sc. degree in the Faculty of Science at University College Dublin. The Undergraduate Program in Pharmacology has 30–35 graduates per year, and approximately half of these students carry on as M.Sc. or Ph.D. postgraduates in the biomedical sciences. This article describes the neuromuscular section of the program, which covers overall 11 different topics. Students entering this course have basic knowledge of muscle biology due to an introductory series of 2nd-year lectures on muscle structure, muscle contraction, muscle reflexes, bioenergetics, ion channel biochemistry, and the physiological basis of ion homeostasis, membrane potential, action potential, signal transduction, and chemical transmission processes.

The course is divided into 10 individual lectures on basic and applied myology. Tutorials to smaller groups of students cover the material of five lectures, and undergraduates are also individually advised on specific aspects of the course. We would like the students to be active participants in the learning process rather than assimilators of large quantities of information. As biomedical information rapidly accumulates in intimidating quantities, it is important to present the essential principles of the specific subject. First we focus our teaching on well established hypotheses, which are discussed in depth at tutorial sessions. Students are then asked to locate and use information sources (such as review articles, research papers, and Internet literature search engines) independently. Groups of three students are given special topics for more in-depth surveys, e.g. the feasibility and ethics of gene therapy, technical difficulties of myoblast transfer therapy, or the medical potential of muscle stem cells. After 2–3 weeks of preparation time, these topics are presented and discussed in small groups. This process helps in the development of independent thinking and encourages students to question and interpret scientific hypotheses and research findings.

To develop the practical skills of students, four laboratory sessions are associated with the lecture series: (i) histological examination of various muscle preparations to understand the fine structure of fibers, (ii) phrenic rat nerve preparation to evaluate the effect of classical agonists and antagonists at the neuromuscular junction, (iii) receptor biochemistry to introduce students to the concepts of tissue homogenization, subcellular fractionation, protein assays, gel electrophoresis, electrophoretic transfer, immunoblot analysis, and the enhanced chemiluminescence detection technique, and (iv) a computer-based practical on the structural analysis of receptors and an overview of structure-function relationships. During their practical thesis work, a group of three to four 4th-year students are exposed to major biochemical techniques used in the biochemical analysis of large membrane assemblies, i.e. differential co-immunoprecipitation, chemical cross-linking, and blot overlay assays with purified and peroxidase-conjugated muscle proteins.

New research findings often lead to the development of a new subdiscipline within a short time; thus it is necessary to exclude some material as well as add material to undergraduate courses. A few new biochemical discoveries may transform biological principles. In this regard, textbooks only provide an introduction into the various aspects of a scientific discipline. To fill the void in the information accessible to students, we include new techniques and developments in advanced lectures. Referring to an article on a new scientific development encourages students to conduct an Internet search on the subject. Supplementing textbook knowledge with information from recent research articles and reviews is essential to providing a stimulating teaching environment. A course on any modern scientific topic represents a survey of currently valid findings. It is also essential to emphasize the significance of experimental design and the logical rationale behind testing hypotheses and to stress that biomedical phenomena can and should be studied at various levels, i.e. populations, whole organisms, organ systems, and isolated tissues and organs as well as at the cellular and molecular level. Only an integrative approach using many different analytical techniques can give a good understanding of biological phenomena.

At the beginning of the advanced course on muscle diseases important aspects of the 2nd-year lectures are recapitulated. The concept of muscle as a biochemical transducer that converts chemical energy into mechanical movement is included. Skeletal muscle is the most abundant tissue in the human body and is not only important for co-ordinated movements and posture but also plays a critical role in body heat homeostasis. Discussed are four major functional characteristics of muscle fibers, i.e. contractility, excitability, extensibility, and elasticity. Special sections of lectures focus on biochemical and biophysical aspects of the sliding filament hypothesis, the innervation and blood supply of muscle fibers, the various layers of connective tissue associated with muscle cells, and the biochemistry of the neuromuscular junction. Light and electron micrographs of transverse and longitudinal sections from developing, mature, aging, and diseased fibers are shown to illustrate the basic arrangement of the contractile apparatus and the extensive internal membrane systems under varying physiological and pathophysiological conditions. The cell biological features of differently sized and shaped human muscles are compared, and special types of muscle as found in the diaphragm or extraocular region are considered.

It is important to stress that an enormous diversity not only exists in the physiological fine regulation of excitation-contraction coupling, relaxation, ion homeostasis, and energy metabolism between the main types of muscle, i.e. skeletal muscle, heart muscle, smooth muscle, and myoepithelial cells, but also between the many subtypes of skeletal muscle fibers. Separate groups of skeletal muscles differ in their cellular heterogeneity in fiber types. This is reflected by distinct variations on the molecular level of individual muscle cells, i.e. differences in levels and expression of muscle-specific protein isoforms. Physiologically these variations in isoform expression levels reflect distinct biological adaptations to specific functional demands. Major biochemical and cell biological topics covered in our muscle biology course are: classes of muscle cell types; skeletal muscle fiber types; ultrastructure of muscle fiber; muscle innervation and blood supply; myogenesis, maturation, and muscle aging; contractile mechanisms; the sliding filament hypothesis; muscle bioenergetics; excitation-contraction coupling; muscle relaxation; muscle ion homeostasis; drugs affecting motor function; the neuromuscular junction; muscle-specific protein isoforms; and molecular myology techniques. A recommended reading list for 4th-year students includes manuscripts on the following topics: muscle structure [1], organization of muscle membrane systems [2], malignant hyperthermia and related disorders of excitation-contraction coupling [3, 4], myasthenia gravis and the nicotinic acetylcholine receptor [5, 6], Duchenne muscular dystrophy and the dystrophin-glycoprotein complex [7, 8], myoblast transfer therapy [9], gene therapy [10], and experimental pharmacological intervention in muscular dystrophy [11].

Preceding the outline of pathobiochemical concepts, an introduction into major biochemical, physiological, molecular biological, and pharmacological methodology used in modern muscle research is given. This includes the measurements of intracellular ion concentrations in muscle cells, the investigation of the movement of ions through channel complexes by the patch clamping technique, and planar lipid bilayers. Major biochemical techniques used in protein purification are reviewed such as subcellular fractionation, protein solubilization, protein assays, protein stabilization, and protection against proteolytic degradation as well as standard separation techniques such as ion exchange chromatography, gel filtration, affinity chromatography, and preparative electrophoresis. For protein identification, students are introduced to the concepts of analytical techniques such as two-dimensional electrophoresis, immunoblotting, N-terminal sequencing, and mass spectroscopy of digested peptide fragments. Pharmacological techniques such as affinity chromatography using immobilized drugs as ligands and standard drug binding assays and screening procedures for novel therapeutic targets are also discussed. Because the important role of modern molecular biological methods is covered in other specialized courses, these techniques are only briefly covered with a review of major recombinant DNA techniques and a description of how gene knock-out and transgenic animals are bred. Because a central aspect of this course focuses on the biochemistry of supramolecular membrane assemblies involved in the molecular pathogenesis of neuromuscular disorders, key techniques used in the investigation of large membrane complexes are reviewed. Established methods of protein chemistry and membrane biochemistry are described. This includes a detailed description of how chemical cross-linking analysis, differential co-immunoprecipitation, domain binding experiments, and various electron microscopical techniques can identify highly complex protein-protein interactions between integral membrane proteins.

Prior to outlining the proposed molecular mechanisms leading to specific muscle disorders, an overview of clinical features and primary molecular defects of common neuromuscular diseases is given. The introductory lecture is not exhaustive but lists selected cellular defects triggered by relatively well established pathobiochemical pathways. Channelopathies, muscular dystrophies, and muscular atrophies, as listed in Table I, are mostly discussed. The description of typical channelopathies includes diseases involving the dihydropyridine receptor, the ryanodine receptor, sodium channels, chloride channels, and potassium channels. Besides genetic skeletal muscle disorders, autoimmune and genetic abnormalities triggered by primary defects in ion channels of the neuromuscular junction are also mentioned. Muscular dystrophies are reviewed according to their primary defects such as mutations in dystrophin, laminin, sarcoglycans, calpain, protein kinase, or emerin. The analysis of muscular atrophies is restricted to two examples, spinal muscular atrophy and Charcot-Marie-Tooth disease. Classical metabolic myopathies are discussed according to specific defects in enzymes.

In the final lecture in this annual course a survey of novel genetic, cellular, and pharmacological approaches to treat neuromuscular diseases is provided. This includes an updated list of newly published research and clinical studies as well as reviews on the molecular medicine of channelopathies, muscular dystrophies, muscular atrophies, and metabolic myopathies. As an example of future directions, the experimental treatment of Duchenne muscular dystrophy is discussed in detail. Potential sites for genetic, cell biological, and pharmacological interventions at different steps of the disease process are identified. Experimental approaches to design new therapeutic strategies such as gene and myoblast transfer therapy are evaluated.

To assess the quality of teaching outcomes, questionnaires are distributed to students at the end of every course. The results of these assessments are summarized and discussed at staff meetings. Student questionnaires often yield significant information on the reception and success of individual courses and are an effective means of improving teaching materials and approaches. Most importantly, discussion with individual students or small groups at tutorials provides useful input for improving next year's lecture material and delivery. Looking at student examination papers for this course it has become clear that many goals of this course have been successful. (i) Students know more about modern biochemical, molecular biological, and pharmacological methodology used in the study of neuromuscular diseases. (ii) Undergraduate science students have been introduced to the key muscle elements involved in ion regulation and membrane stabilization and their role in the pathobiochemistry of selected muscle diseases. (iii) Students appreciate the need to better understand the cause and molecular mechanisms involved in specific diseases to improve treatment strategies. (iv) Students recognize potential sites for genetic, cell biological, and pharmacological interventions during progressive stages of the disease process. This should help future biomedical researchers and health care managers to exploit this knowledge in developing more accurate diagnostic tests and most importantly in evaluating novel attempts at treating devastating human disorders.