Muscle Cell Research Group
Our research
Single muscle fibre (left) being separated from a bundle of muscle fibres (right). The bundle remains intact with the whole EDL muscle (top). The muscle and all fibres remain under paraffin oil and the separation of bundles and fibres is performed using a bifocal dissecting microscope (Image taken at ?? x magnification).
Overview
Our lab examines aspects of muscle function using mechanically-skinned single fibres. We use fibres predominantly from soleus and EDL muscle of Long-Evans Hooded Rats (Rattus norvegicus) and C57BL\10 mice (Mus musculus), as well as the iliofibularis from Toad (Bufo marinus).
The use of mechanically-skinned fibres has a number of advantages:
- the cytoplasmic environment can be accessed and readily manipulated to study individual factors or combinations of factors
- contractile properties are unchanged compared with an intact fibre
- the sarcoplasmic reticulum (SR) remains intact and functional
- the t-system seals off after skinning to form a separate compartment, resulting in normal excitation-contraction (E-C) coupling
EDL muscle following dissection pinned under paraffin oil ready for dissection
Examination of contractile apparatus
By using correctly buffered solutions, the Ca2+-activation properties (ie. Ca2+ sensitivity and Hill coefficient) remain unchanged in mechanically-skinned fibres compared with intact fibres.
When only the contractile apparatus is being examined, it is possible to remove all other membrane bound structures by destroying their membranes using Triton X-100. Such treatment leaves the contractile apparatus intact whilst ensuring no interference to the measurements being made by membrane structures, such as the SR, mitochondria or the t-system.
Over the years, our lab has examined the effect of various metabolites / agents / factors on Ca2+ sensitivity and maximum Ca2+-activated force. These include:
- ATP, ADP, creatine, glucose-6-phosphate, lactate, pH, Mg2+,
- protease inhibitors
- oxidation & reduction, temperature
Single rat muscle fibre being mechanically skinned
under paraffin oil (fibre diameter ~ 50 μm).
The sarcolemma is being 'peeled' back on the left
hand side, forming a 'cuff' as it is removed up
along the fibre.
Examination of SR properties
Ca2+ handling by the SR involves both Ca2+ release and Ca2+ uptake
Ca2+ release channels are called ryanodine receptors (RyR). The opening of the RyR in skeletal muscle is inhibited by physiological [Mg2+] (~ 1 mM) and millimolar [Ca2+]. Ca2+ from the SR can be examined using solutions containing low Mg2+ and caffeine, which remove the inhibition on the RyR.
The release of Ca2+ from the SR results in the production of force, which we measure with a force transducer.
The fibre relaxes as Ca2+ is taken back up into the SR by the Ca-ATPase pumps. Ca2+ uptake into the SR can be examined by using agonists of the Ca-ATPase such as TBQ (2,5-di(tert-butyl)-1,4-benzohydroquinone)
Over the years, our lab has examined the effect of various metabolites / agents / factors on SR function (Ca2+ release and /or Ca2+-uptake). These include:
- ATP, ADP, lactate, pH, Mg2+
Examination of E-C coupling
Depolarisation induced force response
The t-system forms a separate compartment from the myoplasmic environment. By using a solution containing, amongst other constituents, a high concentration of K+, the membrane can be polarised. To depolarise the membrane the fibre can be transferred from the high K+ solution to a high Na+ solution. The fibre can then be repolarised in the high K+ solution.
Over the years, our lab has examined the effect of various metabolites / agents / factors on the depolarisation induced force responses in mechanically-skinned single fibres. These include:
- ATP, ADP, carnosine, cholesterol, glycogen
- chlorpromazine, H-89 (PKA inhibitor)
Electrical stimulation (Action potential induced force response)
Twitch or tetanic force responses can be initiated in mechanically-skinned fibres by the generation of action potentials (AP) in the sealed t-tubules.
Over the years, our lab has examined the effect of various metabolites / agents / factors on the AP induced force responses in mechanically-skinned single fibres. These include:
- ADP, lactate, pH
- oxidation & redox state
