MUSCLE RESEARCH LABORATORY

Joseph F.Y. Hoh

  

  

RESEARCH

PERSONNEL in 1994 and 1995

Joseph F.Y. Hoh Reader (in-charge) University 1971-
Christine Lucas PhD student (from '93) 1991-
Michael Hsu PhD student (from '93) Fac Med S'ship 1993-
Lucia H.T. Kang PhD student (0.5) 1989-
Louise Sieber BMedSc(Hons) student 1994
Han Qin PhD student (0.6; Assoc. S'visor: B. Morris, 0.4)
(RO from '95)
Fac Med S'ship 1993-
Lynne Turnbull RA (Macquarie/Sydney) (0.5) NHMRC 1993-
Angela Hamilton RA (0.1) Honorary 1993-

Current effective full-time personnel = 4.7

  
Research in the Laboratory is concerned with the physiology, biochemistry, molecular biology and evolutionary biology of skeletal and cardiac muscles.
  
PROJECTS in 1994

Molecular biology of cat jaw muscle genes

Previous work in this Laboratory has shown that jaw-closing muscles of carnivores, primates and bats express a specific isoforms of myosin which make these muscles faster and more powerful than limb muscles. The capacity to express this 'superfast' myosin and other jaw-specific myofibrillar proteins has been shown by transplantation experiments to be intrinsic to jaw-closing muscles. The most exciting aspect of the jaw muscle problem is the question of how jaw-specific muscle genes are regulated differently from limb muscle genes. In order to study the regulation of jaw-specific muscle genes, the complete sequence of the MyLC2 gene and much of the very large superfast myosin heavy chain (MyHC) gene has been elucidated. During the year, a 20 kb clone has been isolated from a cat genomic library using a MyLC probe. This clone has now been characterized by partial sequencing; it includes the complete MyLC2 gene and its immediate 5'- and 3'-flanking regions. The MyLC2 gene is composed of 7 exons ranging in size from 20 to 165 bp and span approximately 4 kb of genomic DNA. The 4.8 kb of the currently available superfast MyHC cDNA sequence encodes half of the myosin head or S1 subfragment, which contains the ATPase and actin-binding sites, and the whole of the rod portion of the molecule. The encoded amino acid sequence showed a general low level of homology with other striated muscle MyHC isoforms, implicating its ancient evolutionary origin. The sequence also revealed specific sites which deviated markedly from other MyHCs, which may have specific functionally significant for superfast myosin. These sites including 1) an actin-binding site which has been implicated in controlling myosin ATPase activity, 2) MyLC binding regions, 3) the amino terminal of the myosin rod 4) the hinge region which permits the crossbridge to swing out from the thick filament and 5) a 16-residue insertion near the carboxyl terminal of the molecule.

Skeletal myosin isoforms in marsupial mammals

While skeletal muscle fibre types in eutherian mammals have been extensively investigated, there is no information on fibre types in marsupial mammals. In 1994, a start was made to investigate the marsupial muscle fibre types using immunohistochemical and biochemical methods. The focus of the initial work is how jaw muscles in marsupial carnivores (dasyurids) and herbivores (macropodids) adapt to their lifestyle of these animals. The speed and power of superfast fibres represent an excellent adaptation to the carnivorous lifestyle of carnivores. The divergence in structure of superfast myosin from other myosins suggests that superfast muscle originated very early during vertebrate evolution, possibly dating back to the origin of the vertebrate jaw. It is proposed that the common ancestor of mammals expressed superfast myosin in its jaw muscle, and that during the mammalian radiation herbivores acquired the capacity to express slow myosin which is more appropriate for continuous chewing because of its tension economy. Is superfast myosin expressed in jaw muscles of marsupial carnivores? The answer is a resounding yes! Using a battery of monoclonal antibodies the Lab. raised against cat superfast MyHC, and found that about half of these reacted against jaw, but not limb, muscles of two species of marsupial carnivores, the fat-tailed dunnart and the brown antechinus.

It is widely assumed that eutherian herbivores express slow myosin (b-cardiac) in their jaw muscles. The Lab showed that myosin in jaw muscles of several members of the macropodid family differs from all myosins in the limb muscle fibres, including that of slow fibres. Biochemical evidence suggest that it may be a-cardiac myosin, but this awaits immunochemical verification. This finding was of considerable interest because a-cardiac myosin is known to be expressed in rabbit jaw muscle, and previous work has shown that myosin in slow jaw fibres of the cat and sheep differs from that in their limb slow fibres. It may emerge that 'slow fibres' in all mammalian jaw muscles express a-cardiac myosin. If so, the capacity to express a-cardiac myosin will emerge as a universal character of jaw muscle cells.

The immunohistochemistry of extraocular and eyelid muscles

Extraocular (EO) muscles contract faster than limb fast muscle, the higher speed is associated with the expression of EO-specific fast myosin heavy chains (MyHCs). The levator palpabrae superioris (LPS), an eyelid muscle responsible for elevating the upper eyelid, is phylogenetically a latecomer, being present in mammals but not in birds and reptiles. It evolved by separating from the superior rectus muscle, from which it splits during embryogenesis. Controversy exists over whether the MyHCs expressed in the LPS are limb-like or EO-like. The retractor bulbi (RB) is another accessory EO muscle. It develops from the lateral rectus and its fibre type composition is also controversial. Monoclonal antibodies (mabs) were raised against the EO specific fast MyHC and used together with mabs against limb fast and slow MyHCs to study immunohistochemically the types of MyHCs expressed in fibres of rabbit LPS and RB muscles using HRP-linked indirect immunohistochemistry. Results revealed that the staining pattern of the LPS is different from that seen in limb muscle, but resembles that of the global region of the superior rectus and other EO muscles. Mab 5-4D, which stains limb slow fibres, stains scattered fibres in the LPS, the vast majority of LPS fibres are thus fast. Mab 5-2B, which stains all limb fast fibres, stains only a small subpopulation of small diameter fibres in the LPS. The remaining large population of large diameter fast fibres stains with EO fast specific mabs 4A6, 10A10, 5E2 and 7F8 which do not stain limb muscle fibres. RB muscles gave similar results. Since the EO-specific antibodies recognize at least 2 distinct MyHCs, both isoforms are present in these accessory muscles. Thus, the expression of MyHCs in LPS and RB muscle fibres appears to reflect that seen in fibres of the muscles from which they evolved.

The mechanisms of action of inotropic agents

Endothelin, the potent vasoconstrictor secreted by endothelial cells, has a positive inotropic action on the heart. Previous work revealed that this agent has no action of the dynamic stiffness parameter fmin of the heart, implying that it has no effect on the kinetics of crossbridge cycling. The enhanced force production in the absence of elevation of intracellular Ca2+ suggests that Ca2+ sensitivity is enhanced. These features of the action of endothelin resemble the effects of repetitive stimulation on skeletal muscle twitch force, which is attributable to the phosphorylation of MyLC2. Could the inotropic action of endothelin be due to phosphorylation of cardiac MyLC2? In skeletal muscle, MyLC2 is phosphorylated by myosin light chain kinase, which is activated by the elevated Ca during tetanic stimulation. The level of this enzyme in cardiac muscle is low, but recently it has been shown that, in vitro, protein kinase C, which is known to be activated by endothelin in cardiac muscle, can phosphorylate MyLC2 at the same sites phosphorylated by myosin light chain kinase. In collaboration with Russell Ludowyke, it was shown that endothelin increases the phosphorylation of MyLC2 in papillary muscles equilibrated with 32Pi, consistent with the hypothesis that the inotropic action of endothelin is mediated by protein kinase C via phosphorylation of MyLC2.

The Lab had previously shown that cross-bridge kinetics in rat papillary is enhanced by b-adrenergic stimulation, a cAMP analogue or an inhibitor of phosphodiesterase, isobutyrylmethylxanthine (IBMX), as indicated by an increase in dynamic stiffness parameter fmin. The molecular basis of this action is not yet known, but is likely to be due to the protein kinase A mediated phosphorylation of one of the two candidate myofibrillar proteins: troponin-I and C-protein. It has not been possible to separate the phosphorylation state of these proteins until recently, when it was shown that treating myocytes with the chemical phosphatase 2,3-butanedione monoxime (BDM) led to the dephosphorylation of C-protein without significantly affecting the state of phosphorylation of troponin-I. It was shown that BDM, while reducing rat papillary muscle force, does not affect fmin. Furthermore, in the presence of BDM, the papillary muscle could still respond to IBMX by an increase in fmin. The result favours the notion that enhancement of crossbridge kinetics is due to the phosphorylation of troponin-I. The Lab will attempt to confirm this by biochemical analysis in 1995.
  

RESEARCH PLANNED for 1995

In 1995, sequencing of the superfast MyHC and the MyLC2 gene will continue. The 5'-flanking region and of the MyLC2 gene will be used in transfection assays in order to define the regions of the gene necessary for jaw-specific expression. The MyHC composition of EO muscles will be characterized using SDS gel electrophoresis and western blotting using monoclonal antibodies against extraocular MyHCs. Jaw muscles of eutherian and marsupial mammals as well as lower vertebrates will be studied in order to trace the evolutionary history of superfast and a-cardiac myosins. The actions of various inotropic agents on crossbridge kinetics will continue to be analyzed.
  

COLLABORATIONS

Cardiac myofibrillar protein phosphorylation and cardiac mechanics:
Dr Günther Rossmanith, School of Mathematics, Physics, Computing & Electronics, Macquarie Univ. (1993-present).

Cardiac myofibrillar protein phosphorylation and cardiac mechanics:
Dr Russel Ludowyke, Centre for Immunology, St Vincent's Hospital, Sydney (1994).
  

FACILITIES

The Laboratory occupies rooms 356A, 356B, 356C, 356E and 356F of the Anderson Stuart Building. Dr Hoh's office is room 356D. Facilities exist for tissue culture, development of monoclonal antibodies, immunohistochemistry and histochemistry, protein chemistry and gel electrophoresis. Biochemical equipment includes a Beckman preparative ultracentrifuge with swing-bucket and fixed-angle rotors, Gilford spectrophotometer with linear transport for scanning gels, column chromatography equipment, refrigerated cabinet, radiometer pH meter and pH stat, various electrophoresis apparatus and accessories, including power supplies and apparatus for DNA sequence analysis, thermostatic baths, rotary evaporator, and tissue homogenizer. The tissue culture facility includes two laminar flow cabinets, CO2 incubator, inverted microscope, fluorescence and visible light research microscopes with differential interference contrast and photo-micrographic attachments. Physiological equipment includes digitometer, stimulators, force transducers, temperature regulating unit and storage oscilloscope. A facility exists for analyzing the mechanical properties of single muscle fibres and incorporates a Cambridge Technology 300S ergometer, series 400A force transducer and Keithly data acquisition system for interfacing with NEC APC IV microcomputer. Room 356F is an authorized C1 laboratory for doing recombinant DNA work.
  

OTHER RESEARCH ACTIVITIES

Editorial board of scientific journal

Basic and Applied Myology.

Refereeing

Manuscripts: for Journal of Molecular and Cellular Cardiology (1), Circulation Research (3), Clinical and Experimental Pharmacology and Physiology (2), Acta Physiologica Scandinavica (1).

Grant applications: for NHMRC (7), NHF (2), ARC (2), Health Research Council of NZ (1), NZ Lottery Grants Board (1).

PhD thesis examined: for Univ. of Sydney.

Fellowship applications: for NHMRC (1), ARC (2).

News media

Effect of spaceflight on muscle was featured in the ABC TV program Quantum (repeat broadcast in Feb 1994).

Overseas conferences attended

Keystone Symposium on Molecular Biology of Muscle Development, Snowbird, Utah, USA (Apr)

International Workshop on Sarcomeric Myosins: Structure, Function and Dysfunction. Venice, Italy (Sep).

Seminars

Overseas

Dept of Genetics, Harvard Univ. Medical School, Boston (Oct).

Dept of Ophthalmology, Univ. of Kentucky School of Medicine, Lexington (Oct).

Dept of Physiology, Univ. of Illinois School of Medicine, Chicago (Oct).

Dept of Medicine, Stanford Univ. Medical School, San Francisco (Oct).

Dept of Anatomy and Cell Biology, Univ. of California at San Francisco, San Francisco (Oct).
  

THESIS PASSED in 1994

BMedSc(Hons)

Sieber LG (1994) Evolution of vertebrate jaw muscle. (Result: 2nd Class, Div. 1)
  

5-YEAR RESEARCH PUBLICATIONS

MOST RECENT TOTAL ANNUAL CITATIONS (for 1993): 116

  
FUNDING for 1994 and 1995

NHMRC Analysis of cat jaw muscle cells
using antibodies and cDNA probes
Hoh JFY 1993
1994 $50,809
1995 $51,622

NHMRC Cardiac myofibrillar protein phosphorylation
and cardiac mechanics
Rossmanith GH 1993
Hoh JFY 1994 $35,662

Total for 1994: $50,809 (admin. by Univ. of Sydney only)

Total for 1995: $51,622 (admin. by Univ. of Sydney only)

  

  

TEACHING

COURSES TAUGHT

Medicine 2

Lectures: 3, on regulation of cardiac contractility in the Distinction course.

Practical classes: 2: 1, of 3 h, presented to 4 groups; 1, of 1 h presented to 2 groups; both on skeletal muscle contraction.

Tutorials: 1, presented to 2 groups, on skeletal muscle.

Assessment: Essay written in students' own time.

Medical Science 3/Science 3: Cardiovascular

Course supervisor for Science 3 and co-ordinator for Cardiovascular Function in Sport and Disease option in 3rd year of the Bachelor of Medical Sciences degree.

Lectures: 9: 7 on cardiac muscle, 2 on skeletal muscle.

Practical classes: 2, of 6 h, each presented twice, on cardiac contractility.

Excursions: 3, each of 3 h, to Telectronics, King George V Hospital and Prince of Wales Medical Research Institute respectively.

Tutorials: 10, in which students from small groups presented published scientific papers.

Examination: Choice of essay type questions.

Dentistry 2

Lectures: 6, on muscles, autonomic nervous system and pathophysiology.

Practical classes: 2: 1 of 3 h, presented to 4 groups, on skeletal muscle contraction; 1 of 1 h, presented to 4 groups, on skeletal muscle contraction.

Tutorials: 2: 1 to the whole year; and another presented 4 times to small groups.
  

TEACHING HOURS in 1994

Dent 2 Med 2 Sc3/HLS3 Total
Lectures 6 3 9 18
Practical classes 16 14 24 54
Tutorials 5 2 10 17
Excursions - - 9 9
Lecture preparation 12 12 30 54
Student consultations 3 2 4 9
Marking essays & assignments - 10 24 34
Setting exams 5 - 1 6
Marking exams 7 - 10 17
Marking theses (Hons) - - - 24
Course-supervisor duties - - 60 60

Total formal contact teaching time = 98 h

Total time = 302 h

  

  

ADMINISTRATION

(see also OTHER RESEARCH ACTIVITIES in 1994)

Faculty of Medicine

Member, Executive Committee on Admissions Procedure for the postgraduate degree.

Member, Musculoskeletal Sciences Planning Team for the postgraduate curriculum.