04/25/07



Protein Differences in Muscles and Related Effects

On Their Mechanical Properties

Ludovic Vincent

04/25/07

Background

Muscles serve as the motors for locomotion and movement in mammals. Their machinery is made up of molecular filaments like actin whose organization drives cell shape and whose role serves to resist forces in tension2. Muscle types differ in their actin organization based on their function: skeletal (striated) muscle has complex and highly organized sacromere fibers while smooth muscle has irregularly packed filaments1. Myosin, a molecular motor protein that acts on actin, pulls actin filaments close together during muscle contraction via sliding and cross-linking of myosin and actin filaments3,5 (Plate 1). It was previously found by gel electrophoresis and image analysis that myosin content in the cardiac muscle of rats was significantly higher than in their smooth (gut) muscle (Fig. 1). These results were in accordance Cohen and Murphy’s findings; there is less myosin in smooth muscle than in striated muscle2. Thus, it was shown that muscles that differ in function differ in relative myosin concentration.

Since the structural behavior of tissues depends on their material properties and therefore their protein composition, skeletal muscle is expected to exhibit greater mechanical properties than smooth muscle under uniaxial loading4, 6 (Fig. 3). Gel electrophoresis and image analysis (Exp. 1) will reveal the abundance of actin in skeletal and smooth muscle (Figure 1). In combination, the mechanical properties of the material samples can be determined by loading the muscle uniaxially (Exp. 3) using the Instron 4444 by recording force and displacement over time for a given loading rate using LabView and plotting a stress-strain relationship (Figure 2a, 2b).

Objectives

Hypothesis: Striated muscle abundant in myosin will exhibit greater ultimate strength under uniaxial loading than smooth muscle as a result of protein composition.

A. Experimental Goals

1. To quantify the quantity of muscle motor proteins (myosin in particular) in mammalian muscle using gel electrophoresis and image analysis.

2. To determine if the mechanical strength (ultimate strength in particular) of various muscle types loaded uniaxially depends on the chemical composition of the tissues.

B. Educational Goals

1. Integrate gel electrophoresis experience and knowledge to identify specific proteins through image analysis.

2. Understand how proteins and related biochemical functions affect cell mechanical properties.

3. Apply bioengineering concepts of mechanical and material properties to biological processes.

Equipment

Major Equipment

• Instron Model 4444 benchtop materials testing machine – to uniaxially load the muscle samples.

• BioRad Mini Protean II Cell Electrophoresis System – apparatus needed in order to run the gel needed to quantify the amount of myosin in both skeletal and smooth muscle.

• Power Supply (BioRad PowerPac Basic & 300) – to supply the voltage that will separate the negatively SDS-charged proteins on the Polyacrylamide gel.

Lab Equipment

• Scalpel, scissors, cutting board – prepare muscle samples

• Length measuring instruments: calipers and rulers – measure dimensions of samples

• Heating block – to help the DTT in reducing the protein samples

• Computer with LabView – to measure and record the physical force and displacement measurements outputted by the Instron machine.

Supplies

• Skeletal muscle samples – to use as testing material for the Instron.

• Smooth muscle samples – to use as testing material for the Instron.

• 1x 4-15% ReadyGel Polyacrylamide gel

• 50mL 20x SDS buffer

• 250μL Loading Buffer

• 1x 1.5mL microfuge tubes with DTT

• 1x 1.5mL microfuge tubes

• 10μL SDS-Page Molecular Weight Standards

• 50mL Coomassie Blue Stain

• 50mL Destain solution

• 1x Plastic container

• Pipettes and tips (regular and gel-loading)

• Safety Gloves

• Safety Glasses

Newly Purchased Equipment

• Meat Slicer – In order to have samples of similar dimensions, the striated and smooth muscle must be cut consistently. Cutting the samples in similar lengths and height can be done with the use of a scalpel, knife, scissors, and a ruler. However, obtaining samples that have equal thicknesses is not an easy task by hand. A meat slicer will facilitate this process, providing sheets of muscle of equal breadth. Those slices, or slabs, will then be cut by hand to the desired surrogate dimensions.

• Small sponge – to more effectively absorb Coomassie blue staining solution when gel is in destain. Real gels are not destained using paper towels in real labs.

Proposed Methods and Analysis

A. Protocol

|1) |Two members prepare the muscle tissues for gel electrophoresis as described in Exp. 1 protocol, page 9-10, section A, |30min |

| |B1, B2. | |

|2) |Load 20μL of the loading buffer rich in skeletal muscle proteins in lanes 1-4 and smooth muscle containing buffer in |05min |

| |lanes 6-9. Load 10μL of ladder into lane 5. | |

|3) |Run the gel for 35min at 100V. |35min |

|4) |Meanwhile, other two members are preparing muscle samples cutting 6 samples of each type of muscle. Dimensions |70min |

| |1cm*4cm*0.5cm. Sample should first be cut in thin slices using the meat slicer (0.5cm) and then by hand using a scalpel | |

| |to the desired dimensions. Store samples on wet paper towels. | |

|5) |Put gel in 50mL stain solution (Coomassie Blue) in plastic container for 1 hour. |60min |

|6) |While gel is staining, load a Smooth muscle sample on Instron as described in Exp. 3 protocol, page 5-6, section B. |10min |

| |Measure the thickness, height, and width of the sample with calipers after it has been loaded. These are your true | |

| |specimen dimensions. | |

|7) |Record the data in a separate excel file using LabView. Load the sample at 5mm/min recording 20pts/sec. Repeat for other|50min |

| |five samples. | |

|8) |Take gel out of staining solution and carefully place in 50mL destain solution with small sponge (cut one big sponge in |120min |

| |4 small sponges). Replace the sponge after one hour with a new one. | |

|9) |Repeat steps 5 and 6 using the six Skeletal muscle samples this time. |60min |

|10) |Clean Instron machine, properly dispose of samples, solutions, and pipette tips. |30min |

|11) |Take a picture of the gel with Sharp CCD camera. Exp. 1, page 12, section F |10min |

| | | |

| |Total Experiment time – 4h20min/3h40min |

Note: Italicized steps should be done at the same time as non-italicized steps as the two parts of this experiment do not depend on each other and can be performed simultaneously. This requires the group to split into two groups of twos for part of the experiment. While the gel is running, staining, and de-staining, the entire group can work together as these steps require little attention as long as the group members monitor the time correcty.

B. Analysis

Run MatLab and the m-file gelanalysisv2.m provided in the BE210 folder. Using the imaging software, pick a region of interest (ROI) around the band at 200kDa. Pay special attention to incorporate the entirety of the band within your ROI without selecting neighboring bands. Remember, the first band on the molecular marker gel is the myosin heavy chain. Since this is the protein of interest for this lab, the myosin present in smooth and skeletal muscle should appear along the same line as that of the marker. Repeat this multiple times (n=5) for accuracy of individual band quantification. Relate the average pixel value above threshold for each lane to the standard marker (information about protein abundance in the marker can be found on Blackboard in the Experiment 1 folder). For each muscle, tabulate averages (in μg of protein loaded) and standard deviations for each lane and for the four lanes combined. Perform a one tailed t-test assuming equal variance for the set of the eight averages, testing for a significantly greater amount of myosin in skeletal muscle over smooth muscle.

Tabulate sample geometry for the uniaxially loaded samples. Include cross sectional area, width, height, and thickness. Write a Matlab program that calculates the stress and strain for all the data points of all 12 samples (Hint: use Equation 1 and 2). Still in Matlab, plot the stress versus strain relationship for each surrogate. Using the maximum function, determine the ultimate strength of the material. Tabulate the 12 ultimate strengths in a table below your graph. Perform a one tailed t-test assuming equal variance for the set of the 12 averages, testing for a significant increase in ultimate strength for the skeletal muscle.

If there is both a significant increase in failure strength and myosin abundance in striated muscle over smooth muscle, then this provides evidence that supports the hypothesis. If there is a significant increase in myosin composition in skeletal muscle over smooth muscle but no significant difference in failure strength between the two muscles, then the hypothesis is disproved.

Potential Pitfalls & Alternative Methods/Analysis

Previous experiments showed that there was a significant increase in myosin content in skeletal compared to smooth muscle. If the gel has not destained sufficiently in the allotted time, there might not be enough contrast between the protein bands and the dyed polyacrylamide for the Matlab program to effectively determine pixel values above the background threshold. In this case, the user can run the program by analyzing the light chains of actin as there are two myosin light chains (M.W. ~20,000Da) for every heavy chain (M.W. ~200kDa)1. In theory, the protein band of myosin light chains should be twice as intense as the heavy chain band because of the 2:1 stoichiometric ratio. Consequently, the one tailed t-test assuming equal variance between both types of muscle can be carried out.

However, there potentially exist many proteins, and therefore protein bands on the gel, whose molecular weight ranges between 15 and 25kDa. Thus, the threshold value (set by the borders of the box in the Matlab program) may overlap onto a an existing protein band near the myosin light chain.

Budget

$279.95 Electric Food Slicer - #632.

Maker: Chef'sChoice®-International, VariTilt Slicer

Specifications: 100W motor, gravity feed recliner up to 30deg, gear drive, large 7in (approx. 17cm) diameter multi-purpose stainless steel blade, cantilevered design convenient for larger trays and easy clean-up, precision thickness control from deli-thin to 5/8 inch, retractable food carriage. Key parts are constructed of cast aluminum and stainless steel and rubber feet hold the slicer firmly to the work surface for stability.

$25.00 2lbs Skeletal Muscle – Ranch Foods Direct, New York Striploin @ $12.49/lbs

Specifications: Colorado Springs cows, grain-finished on a diet rich in good fats and high in Vitamin E.

$04.00 1x rat stomach – Provided by Dr. Brittany Coats’ favorite slaughter house

$06.00 3x O-Cel-O Sponges, Handy Size (Colors May Vary) 4 ea @ $1.99

Parent Company: 3M

Specifications: 119mm x 76mm x 15mm (1 sponge)

$314.95 Total

Appendix

[pic]

Fig. 3 shows the impacts of axial loading

on aortic arties (smooth muscle) of dogs.

Figure 2a(left) and 2b(right) - Figure 2a shows the Force vs. Displacement graph and figure 2b shows the stress vs. strain graph of each of the 5 mm/min and 100 mm/min trials

Equation 1: σ = F/A = F/(w*t)

Equation 2: ε = d/L

σ = stress (in Pa), F = force (in Newtons), A = area (in m2), w = width (in meters), t = thickness (in meters), ε = strain, d = change in height of sample (in mm), L = length of sample before loading (in mm)

References

1Murray, Robert K. “Actin & Myosin Are the Major Proteins of Muscle.” Access Medicine (2007) 22 April 2007 .

2Cohen, D. M., and Murphy, R. A. (1978) J. Cen. Physiol. 72,369-380

3Harris, D.E., S.S. Work, R.K. Wright Wright, N.R. Alpert, and D.M. Warshaw. “Smooth, Cardiac and Skeletal Muscle Myosin Force and Motion Generation Assessed by Cross-Bridge Mechanical Interactions in Vitro.” Journal of Muscle Research and Cell Motility 15 (1994): 11-19. 28 Feb. 2007 .

4Cox, Robert H. “Three-dimensional mechanics of arterial segments in vitro: methods.” J. Appl. Physiol. 36(3) : 38 l-384. I974,-E

5Elliott GF, Lowy J, Millman BM., “Low-angle x-ray diffraction studies of living striated muscle during contraction.” J Mol Biol. 1967 Apr 14;25(1):31-45

6Elliott GF, Variations of the contractile apparatus in smooth and striated muscles. X-ray diffraction studies at rest and in contraction.

J Gen Physiol. 1967 Jul;50(6):Suppl:171-84.

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