Joseph F.Y. Hoh

RESEARCH in 1993

Molecular biology of cat jaw muscle genes

Previous work in this laboratory has shown that cat jaw-closing muscles express specific isoforms of myosin, tropomyosin and C-protein not found in limb muscles, and that the capacity to express these isoforms of myofibrillar proteins is intrinsic to jaw-closing muscles. The most exciting aspect of the jaw muscle problem is the question of how jaw muscle genes are regulated differently from limb muscle genes. In order to study the regulation of jaw-specific muscle genes, the superfast myosin heavy chain (MyHC) and superfast myosin light chain-2 (MyLC2) genes were cloned by Han Qin. During the year, he elucidated the complete sequence of the MyLC2 gene. A comparison of this sequence with other MyLC2 genes in the Genbank revealed relatively low homology with limb fast and slow MyLC2 but a high homology with a published human MyLC sequence which we identify as a human homologue of the superfast MyLC2. A phylogenetic tree for the MyLC2 gene derived from sequence comparisons (see figure) revealed that superfast myosin is of very ancient origin. The superfast MyHC is a large gene, the full-length cDNA is expected to be about 6 kb long. Sequencing of this gene is still proceeding. About 4.5 kb has been completed, and preliminary sequence comparisons with other MyHC genes also affirm its ancient evolutionary origin.

Evolution of jaw-closing muscles

Superfast muscle fibres are known to be expressed in carnivores and primates, but not in common laboratory and domesticated animals except cats and dogs. The brain structure of flying foxes differs from that of microbats in showing many features found in primates. In collaboration with Jack Pettigrew, attempts were made to resolve the controversy of whether flying foxes should be classified as primates, rather than being placed with the microbats, by examining the fibre types of their jaw muscles. Immunohistochemical analysis using monoclonal antibodies against superfast myosin revealed that superfast fibres were found in representatives of both flying foxes and microbats, with the exception of one species of microbat out of six chiropterans tested. This finding of superfast myosin expression in another major order of placental mammals raises the possibility that superfast fibres may be found in jaw muscles of other mammals, and indeed, in other classes of vertebrates. In view of structural evidence of the ancient origin of the superfast MyHC and MyLC2 described above, the Laboratory has proposed from its research that the superfast fibres first appeared in fish when the jaw first evolved. Superfast myosin has then been expressed by default in carnivorous vertebrates and their close relatives ever since, but animals that have long deviated from the carnivorous lifestyle have evolved mechanisms for expressing other myosins in jaw muscles.

The cell biology of laryngeal and extraocular muscles

Laryngeal and extraocular muscles are highly specialized, with fibre types and myosins not found in limb or jaw muscles. Extraocular muscles are extremely fast contracting, a property shared by all intrinsic laryngeal muscles except the cricothyroid. A definitive fibre type classification for these muscles base on MyHC isoforms such as that presently available for limb and trunk muscles has not been established. In earlier work, the Laboratory resolved five immunohisto-chemically distinct types of extraocular fibres in the cat using a battery of anti-MyHC monoclonal antibodies, but only one of these antibodies (4A6) was extraocular-specific. In 1993 the Laboratory raised another extraocular-specific monoclonal antibody (3A3), which stained a different subset of cat extraocular fibres, suggesting that there are at least 2 distinct MyHCs in extraocular muscles. Unfortunately, neither of these antibodies reacted with SDS-denatured MyHCs, making biochemical characterization of their antigens more difficult. Nevertheless, 3A3 was shown to stain fibres in all the intrinsic laryngeal muscles except the cricothyroid, the fibres of which are indistinguishable immunohisto-chemically from limb muscle fibres. Thus the high speed of contraction of extraocular and laryngeal muscles can be related to the presence of common myosins in these muscles.

Left panel: Phylogenetic tree of myosin light chain-2 gene generated by the 'pileup' programme (GCG) using the superfast MyLC coding sequence and published vertebrate MyLC2 sequences. Right panel: Phylogenetic tree of myosin heavy chain gene based on the terminal 1512 bp of the superfast MyHC sequenceand corresponding published sequences of vertebrate MyHC sequences.

Theginotropic action of endothelin on the rat myocardium

Endothelin, the potent vasoconstrictor secreted by endothelial cells, has a positive inotropic action on the heart. This project sought to determine how the inotropic action of endothelin is implemented at the myofibrillar level. In principle, there are two basic ways to bring about an inotropic action at this level: (i) by increasing the number of active crossbridges and (ii) by increasing the rate at which they turn over ATP. Endothelin is known to increase the number of active crossbridges without increasing diastolic or systolic intracellular Ca2+, suggesting that the Ca-sensitivity of the myofibrillar proteins is enhanced. Certain inotropic agents enhance the kinetics of crossbridge cycling in addition to recruiting more crossbridges. These include b-adrenergic agents and other agents which elevate intracellular cAMP. During the year, the Laboratory asked whether endothelin also increases the crossbridge cycling frequency. Experiments were then carried out which verified that endothelin (100 nM) had an inotropic action on isometric twitches of rat atrial and ventricular tissues. Dynamic stiffness analysis after 30 min incubation of rat ventricular papillary muscle preparations with 100 nM endothelin showed that the stiffness minimum frequency (fmin) derived from these measurements was unaltered, implying that the agent has no action on the rate of crossbridge cycling. The Laboratory next enquired whether endothelin had an effect on crossbridge cycling rate in atrial muscle. As all previous work on dynamic stiffness analysis had been done on ventricular tissue, the fmin of normal rat atrial tissue was first measured. Surprisingly, the fmin turned out to be considerably higher than that for rat papillary muscle. It is known that atrial tissue has the same a cardiac myosin heavy chain as juvenile ventricular tissue, but has atrial-specific myosin light chains. This suggested that the atrial light chains, or some other atrial myofibrillar protein, have a strong influence on crossbridge cycling rate. However, endothelin has also no action on the fmin of atrial muscle. This then led to the conclusion that the inotropic action of endothelin is solely due to crossbridge recruitment, and has no component on crossbridge kinetics.

RESEARCH PLANNED for 1994

In 1994, sequencing of the superfast MyHC will continue. Several cat MyLC2 genomic clones have been isolated from a cat genomic library. The 5'-flanking region of this gene will be sequenced and MyLC2-chloramphenicol acetyltransfer-ase expression vectors constructed for transfection studies in order to define the regions of the gene necessary for jaw-specific expression. More monoclonal antibodies against extraocular MyHCs will be raised and used to characterize extraocular and laryngeal myosins and muscle fibre types. Jaw muscles of lower vertebrates and marsupial animals will be studied in order to trace the evolutionary history of superfast myosin. The actions of various inotropic agents and regulatory proteins on crossbridge kinetics will also be analyzed.

PERSONNEL in 1993 and 1994

Joseph F.Y. Hoh		Reader (in-charge)		University		1971-
Han Qin			PhD student (3/5; Morris 2/5)	Fac. Med. Sch'ship	1989-93
			Research Assistant 		NHMRC 			1993-
Ajita Rughani		Research Assistant I		NHF from 1992		1989-93
Suzanne Hughes		Research Assistant I (2/5) 	NHMRC	1982-93
Angela Hamilton		Research Assistant (1/10)	NHMRC/honorary 	1993-
Christine Lucas		MSc/conv. to PhD student from 1993	1991-
Michael Hsu		MSc student			Fac. Med Sch. '94-	1993-
Lucia H.T. Kang		PhD student			Fac. Med. Sch. to 92 1991-
Effective full-time personnel = 6.5
COLLABORATIONS

Cardiacgmyofibrillar protein phosphorylation and cardiac mechanics: Dr Gunther H. Rossmanith, School of Mathematics, Physics, Computing and Electronics, Macquarie Univ. (1993). Rolegof regulatory proteins on crossbridge kinetics: Dr Gunther H. Rossmanith, School of Mathematics, Physics, Computing and Electronics, Macquarie Univ. and Dr Gregory J. Wilson, Department of Pathology (1993). Musclegfibre types in jaw and limb muscles of flying foxes and microbats: Prof Jack D. Pettigrew, Vision, Touch & Hearing Research Centre, Dept of Physiology, Univ. of Queensland (1993). FACILITIES Theglaboratory 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 photomicrographic 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. FUNDINGgfor 1993 and 1994 NHMRC Regulationgof muscle Hoh JFY 1991 genegexpression 1992 1993 $49,529 Analysisgof cat jaw muscle Hoh JFY 1993 $58,253 cellsgusing antibodies and 1994 $50,809 cDNAgprobes 1995 Cardiacgmyofibrillar protein Rossmanith GH 1993 $35,662 phosphorylationgand cardiac Hoh JFY 1994 $35,662 mechanics, 1995 (Administeredgby Macquarie Univ.) NHF Theginotropic action of Hoh JFY 1992 endothelin-1gon the rat 1993 $37,240 myocardium Totalgfor 1993: $145,022 Totalgfor 1994: $50,809 SCHOLARSHIPS Facultygof Medicine Scholarship Han Qin 1989-93 Facultygof Medicine Scholarship Michael Hsu 1994- TEACHINGgin 1993 Medicineg2 Lectures:g3, on regulation of cardiac contractility in the distinction course. Practicalgclasses: 2: 1, of 3 h, repeated to 4 groups; 1, of 1 h repeated to 8 groups, both on skeletal muscle contraction. Tutorials:g1, repeated to each of 8 groups, on skeletal muscle. Assessment:gBy essay written in students' own time. MedicalgScience: Human Life Sciences 2 Practicalgclasses: 2, of 3 h and 1 h respectively, repeated to 4 groups, on skeletal muscle contraction. Tutorials:g1 to whole class, another, repeated to 4 groups, on skeletal muscle. MedicalgScience 3/Science 3: Cardiovascular Coursegsupervisor for Science 3 and coordinator for Cardiovascular Function in Sport and Disease option in 3rd year of the Bachelor of Medical Sciences degree. Lectures:g8: 7 on cardiac muscle, 1 on skeletal muscle. Practicalgclasses: 2, of 6 h, each repeated once, on cardiac contractility. Tutorials:g6, in which students from small groups present published scientific papers. Examination:gchoice of essay type questions. Dentistryg2 Lectures:g6, on muscles, autonomic nervous system and pathophysiology. Practicalgclasses: 2: 1, of 3 h, repeated to 4 groups, on skeletal muscle contraction, 1, of 1 h, repeated to 2 groups, on skeletal muscle contraction. Tutorials:g1 to the whole year, and another, repeated to 2 small groups. Examination:gshort answer and multiple choice questions (of the 5 alternatives, one correct, variety) Scienceg2 Practicalgclasses: 2: 1, of 3 h, repeated to 2 groups, and 1, of 1 h, repeated to 2 groups, on skeletal muscle contraction. Tutorials:g1. Practicalgclasses: 2, of 5 h and 3 h, respectively, repeated to 3 groups, on skeletal muscle contraction. Tutorials:g1, repeated to 3 groups, on skeletal muscle. Pharmacyg1 Lectures:g5, on muscle, autonomic nervous system and pathophysiology. Examination:gmultiple choice questions (of the 5 alternatives, one correct, variety). Totalgdistribution (hours of formal teaching) Pha1 Dent2 Med2 HLS2 gSci2 Sci3/HLS3 Total Lectures 5 6 3 8 22 Practicalgclasses 16 20 12 4 24 76 Tutorials 2 8 9 1 6 26 Lecturegpreparation 10 12 12 24 58 Studentgconsultations 5 3 2 4 14 Markinggessays 3 24 27 Settinggexams 3 5 1 9 Markinggexams 7 10 17 Markinggtheses 24 (Hons) Course-supervisorgduties 40 40 Totalgformal contact teaching time = 124 h. Totalgtime = 293 h. Other:gMarking Honours thesis = 12 h. OTHERgACTIVITIES in 1993 Editorialgboard of scientific journal Basicgand Applied Myology Refereeing Manuscriptsgfor: Basic and Applied Myology (1), Circulation Research (1). Grantgand Fellowship Applications: for NHMRC (7), ARC (4), Health Research Council of New Zealand (1), New Zealand Neurological Foundation (1). Facultygof Medicine Member,gExecutive Committee on Admissions Procedure for the post-graduate degree Member,gMusculoskeletal Sciences Planning Team for the post-graduate curriculum Member,gPeter Bancroft Prize Committee 5-YEARgRESEARCH PUBLICATIONS JOURNALgARTICLES 1989

HohgJFY, Hughes S (1989) Immunocytochemical analysis of the perinatal development of cat masseter muscle using anti-myosin antibodies. Journal of Muscle Research and Cell Motility, 10, 312-325 HillgMA, Ecob-Prince MS, Hoh JFY (1989) Regeneration of cat posterior temporalis muscle in culture. Cell Differentiation & Development, 28, 145-152 1990

QingH, Kemp J, Yip MY, Lam-Po-Tang PRL, Hoh JFY, Morris BJ (1990) Localization of human cardiac b-myosin heavy chain gene (MYH7) to chromosome 14q12 by in situ hybridization. Cytogenetics and Cell Genetics, 54, 74-76 * 1991

HohgJFY, Rossmanith GH, Hamilton AM (1991) Effects of dibutyryl cyclic AMP, isobutylmethylxanthine and ouabain on crossbridge kinetics in rat cardiac muscle as measured by frequency domain analysis. Circulation Research, 68, 702-713 HohgJFY (1991) Myogenic regulation of mammalian skeletal muscle fibres. News in Physiological Sciences, 6, 1-6 HohgJFY, Hughes S (1991) Expression of superfast myosin in aneural regenerates of cat jaw muscle. Muscle & Nerve, 14, 316-325 HohgJFY, Hughes S (1991) Basal lamina and superfast myosin expression in regenerates of cat jaw muscle. Muscle & Nerve, 14, 398-406 1992

HohgJFY, Hughes S, Walker M., Kang LDH, Everett AW (1991) Slow myosin heavy chains in cat jaw and limb muscles are phenotypically distinct: expression of jaw-specific slow myosin phenotype in regenerated and chronically stimulated jaw muscles. Basic and Applied Myology, 1, 285-294 SinhagAK, Roses RJ, Pozgaj I, Hoh JFY (1992) Indirect myosin immunocyto- chemistry for the identification of fibre types in equine skeletal muscle. Research in Veterinary Science, 53, 25-31 RileygDA, Ellis S, Giometti CS, Hoh JFY, Ilyina-Kakueva EI, Oganov VS, Slocum GR, Bain JLW, Sedlak FR (1992) Muscle sarcomere lesions and thrombosis after spaceflight and suspension unloading. Journal of Applied Physiology, 73, S33-S43 HohgJFY (1992) Muscle fiber types and function. Current Opinion in Rheumatology, 4, 801-808 1993 HohgJFY, Hughes S, Kang LHD, Rughani A, Qin H (1993) The biology of cat jaw-closing muscle cells. Journal of Computer-Assisted Microscopy, 5, 65-70 Earlyg1994 HohgJFY, Wah MSY, Everett AW, Hughes S (1994) Immunocytochemical analysis of skeletal muscle fibers in the toad, Bufo marinus: developmental and seasonal variations on myosin isoform distribution. Basic and Applied Myology (in press) KanggLHD, Hughes S, Pettigrew JD, Hoh JFY (1994) Jaw-specific myosin heavy chain gene expression in sheep, dog, monkey, flying fox and microbat jaw-closing muscles. Basic and Applied Myology (in press) QingH, Morris BJ, Hoh JFY (1994) Isolation and structure of cat superfast myosin light chain-2 cDNA and evidence for the identity of its human homologue. Biochemical and Biophysical Research Communications (in press) * *Indicatesgthat item also appears in list for B.J. Morris CONFERENCEgABSTRACTS AND PRESENTATIONS in 1994 WilsongGJ, Rossmanith GH, Haroothunian S, Hoh JFY (1993) Ca2+-independent force generation in striated muscle. Proceedings of the 58th Meeting of the Australian Physiological and Pharmacological Society, 24, 135P. Melbourne (Sep) RossmanithgGH, Wilson GJ, Haroothunian S, Hoh JFY (1993) Crossbridge dynamics during calcium-independent force generation. Proceedings of the 58th Meeting of the Australian Physiological and Pharmacological Society, 24, 136P. Melbourne (Sep) TurnbullgL, Rossmanith GH, Hoh JFY (1993) Endothelin has no effect on crossbridge kinetics in rat cardiac muscle. Proceedings of the 58th Meeting of the Australian Physiological and Pharmacological Society, 24, 137P. Melbourne (Sep) LucasgCA, Rughani A, Hoh JFY (1993) Immunocytochemical analysis of muscle fibres in cat extraocular and laryngeal muscles. Proceedings of the 12th Annual Meeting of the Australian and New Zealand Society for Cell Biology. Melbourne (Sep) HohgJFY, Qin H (1993) Cloning and Sequencing of the feline superfast myosin light chain-2 gene. Cell Biology International, 17, 802. 12th Annual Meeting of the Australian and New Zealand Society for Cell Biology. Melbourne (Sep)