Muscle Physiology, Cell Biology & Proteomics

Muscle Performance:  
(Jatin G. Burniston and David F. Goldspink)

Functional adaptations in the whole heart and individual skeletal muscles are measured using cardiac power (Figure 1) and oscillatory work loops (Figure 2) in response to:

• ageing,
• exercise interventions,
• anabolic agents (e.g. b₂-agonists) and,
• exposure to excess hormones including; catecholamines, angiotensin II, aldosterone etc.

Mechanistic explanations for these adaptations in muscle function are sought at a cellular and molecular level.

Figure 1 – Cardiac function. Real-time recordings of blood pressure and cardiac output at rest. Cardiac power output (CPO) is the product of mean arterial pressure (MAP) multiplied cardiac output and is expressed as watts (W) after multiplication with the conversion factor (2.22 x 10-3). The subjects reserve capacity (cardiac reserve) is calculated by subtracting CPO at rest from CPO at their VO2max.

Figure 2 – Skeletal muscle function. Work loops progress in a counter-clockwise direction. Hence, positive deflection in position represents an increase in muscle length and ‘negative’ work consumed by the muscle. As the muscle contracts to the shortened position (negative deflection) positive power (work) is produced.

 

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Muscle cell biology:

(Georgina M. Ellison, Jatin G. Burniston and David F. Goldspink)

At the cellular level, we employ techniques of immunohistochemistry, morphometry, microscopy and image analysis to investigate the adaptive changes in the number, size, senescence, renewal (Figure 3) and death (Figure 4) of myocytes in samples of cardiac and skeletal muscle. This enables us to explain changes in whole muscle function.

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Proteomics:

(Jatin G. Burniston)

Changes measured at the whole-muscle and cellular levels are investigated further using proteomics techniques. Data are integrated across each level, i.e. molecular, cellular, whole organ and organism, in order to determine which of the cellular and molecular adaptations best predict a particular change in function.

The Laboratory is equipped with a range of sophisticated proteomics instrumentation, including:
i) a matrix assisted laser desorption ionisation tandem time of flight (MALDI-ToF/ToF) mass spectrometer (Axima TOF2, Shimadzu Biotech),
ii) integrated high performance liquid chromatography system and AccuSpot robot (Shimadzu Biotech),
iii) Xcise digestion robot (Proteome Systems), and
iv) chemical inkjet printer (ChIP, Shimadzu Biotech).
The Laboratory is also equipped with basic items necessary for sample preparation and houses dedicated computing facilities that support image analysis such as Progenesis 100 % same-spot software (Nonlinear Dynamics) for 2D gels and the in-house Mascot server (Matrix Science) used to search mass spectrometry data against protein and genome databases.

A variety of gel-based and gel-free approaches are used to investigate the muscle proteome. For example, cytosolic proteins are routinely separated using two-dimensional gel electrophoresis (2DE; Figure 5).

 

Figure 5 – Separation of cytosolic protein fraction by 2DE. Proteins are separated according to isoelectric point (from left to right) and molecular weight (from top to bottom) and the gel is stained with colloidal Coomassie.

 

Gel images, for instance from sedentary control and exercised-trained muscle, are differentially analysed to determine which protein spots have changed significantly in response to the intervention (Figure 6).

Figure 6 – Differential analysis of 2DE gel spots.

Magnified image of a narrow section of a 2DE gel from approximately 70 kDa to 50 kDa. Insets show three-dimensional reconstructed images of gel spots from sedentary control and exercise-trained muscles. Arrows point to protein spots that were significantly decreased in exercise-trained muscle and later identified as phosphoglucomutase 1. Nominal mass 61,352, calculated pI value: 6.14. gi|77627971.

To identify the proteins, gel spots are excised (Figure 7) and digested into peptides that can be measured using mass spectrometry. The resultant mass spectra (Figure 8) are used to search databases to reveal the identity of the protein and bioinformatic tools are used to interrogate the findings.

Protein spots that differ significantly between experiment groups are cut from the gel and digested into peptides using a Proteome Systems Xcise robot. The movie shows the gel cutting process, which is directed by the image analysis software. Each gel plug is then automatically digested into peptides, which are spotted on to stainless steel plates ready for mass spectrometry.

 

Figure 8 – Mass spectrometric identification of proteins.

Peptide digests are analysed by MALDI-ToF/ToF mass spectrometry to produce a peptide mass fingerprint (A), which is often sufficiently unique to unambiguously identify the parent protein, in this case: Lactate Dehydrogenase A. This identification can be confirmed by using an ion gate to select individual peptide ions, for example mass/charge 1118.81 (B), which are subjected to collision-induced dissociation to create fragment ion spectra (C) indicative of the amino acid sequence of the peptide (K.SADTLWGIQK.E). A Mascot fragment ion search revealed this peptide sequence corresponds to residues 319-328 of Lactate Dehydrogenase A.