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How to plan and execute contractile measurements in permeabilized muscle fibers aurora scientific

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Join Matt Borkowski and Dr. Tim West for this informative webinar covering the A-to-Z of assessing muscle performance and contractile function in single muscle fibers.

For more information: https://insidescientific.com/webinar/how-plan-execute-contractile-measurements-permeabilized-muscle-fibers-aurora-scientific

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How to plan and execute contractile measurements in permeabilized muscle fibers aurora scientific

  1. 1. How to Plan and Execute Contractile Measurements in Permeabilized Muscle Fibers Join Matt Borkowski and Dr. Tim West for this informative webinar covering the A-to-Z of assessing muscle performance and contractile function in single muscle fibers.
  2. 2. Matt Borkowski Sales & Support Manager Aurora Scientific How to Plan and Execute Contractile Measurements in Permeabilized Muscle Fibers Tim West, PhD Chief Technician & Laboratory Manager Royal Veterinary College
  3. 3. InsideScientific is an online educational environment designed for life science researchers. Our goal is to aid in the sharing and distribution of scientific information regarding innovative technologies, protocols, research tools and laboratory services
  4. 4. To access webinar content, Q&A reports, FAQ documents, and information on lab workshops, subscribe to our mail list
  5. 5. Instrumentation and Methodology for Contractile Measurements of Muscle Fibers Matt Borkowski Sales & Support Manager Aurora Scientific Copyright 2019 M. Borkowski, Aurora Scientific and InsideScientific. All Rights Reserved.
  6. 6. About Aurora Scientific Cell Whole Animal Fiber Whole Muscle • Aurora Scientific has served the muscle community for nearly 20 years • Test systems and solutions ranging from single cells up to the whole animal • Friendly, reliable support
  7. 7. • Today we will focus on our permeabilized fiber test system: 1400A • A versatile system for studying the myofilament in skeletal, cardiac or smooth fibers from virtually any species 1400A: Permeabilized Fiber Test System Force – pCa kTR Force- Velocity About Aurora Scientific
  8. 8. • The sarcolemma of the tissue is partially made porous • Typically done by chemical means • The tissue becomes ‘permeable’ to calcium • Contraction will occur when immersed in a high calcium solution Figure courtesy of Saks et al. 1998 What are Permeabilized Fibers?
  9. 9. How can we handle these fibers? • The fibers must be coupled to specially built metal hooks • Usually done either by suturing or with thin metal foil ‘T-clips’ • Strong attachment without compliance is critical to transmit the contractile force to the system accurately Fiber photo courtesy of Claflin lab Mock model of fiber with T-clips (right) Skeletal fiber sutured (left)
  10. 10. Typical Experiments: Force - pCa • Fibers are held at constant length (isometric) • Immersed in bathing solutions of different calcium concentrations • Force data can be normalized to fiber cross sectional area and fit to a hill equation showing sensitivity to calcium Figures from Freidrich et al. Frontiers in Physiology 2016
  11. 11. • Fibers are activated maximally in a high calcium bathing solution • We measure the rate of tension redevelopment following a rapid release and re-stretch in high calcium • Useful for understanding the dynamics of the myofilament and cross bridge cycling Figure from Clay et al. American Journal of Physiology 2015 Typical Experiments: Force - kTR
  12. 12. • Fibers are again activated maximally in a high calcium bathing solution • Through feedback control in software load clamps to a % of maximum are implemented and shortening velocity measured • Useful for quantifying the power output of the tissue Figure from McDonald et al. Biochemistry Research 2012 Typical Experiments Force: Velocity
  13. 13. How Does it Work ?
  14. 14. • Typical transducer choice for fibers has max working range of 5 or 10mN • Force Measurement resolution of 0.1-0.2 uN • Extremely low compliance (less than <5 microns) • Frequency response of 1kHz 403A Force Transducer – 5mN 400A Series - Force Transducer
  15. 15. • Force transducer is based on a capacitive sensor • Unit has an active measurement ‘beam’ as well as a parallel reference beam • Sensor a happy medium between durability/performance versus optical & strain gauge designs • CMMR – Eliminates the need for complicated filtering
  16. 16. 322C - High-Speed Length Controller • Long working range (maximum 6mm) • Very fast step response time (<0.5ms) • Frequency response in excess of 2kHz • Can sustain tensile loads up to 300mN 322C – High Speed Length Controller
  17. 17. • Length Controller is based upon a fast servo motor element • Allows large stretches and slacks with closed loop position control which can not be accomplished with most piezo elements • Rotational movement can be approximated as linear for the small angular excursions
  18. 18. • Automated 8 well plate with optional 120 or 160 uL volumes • Temperature control via peltier module between 0-40 degrees Celsius • Complete bath to bath index time of 1.2s 802D apparatus shown with 403A and 322C 802D - Permeabilized Fiber Apparatus
  19. 19. • Designed to work with most inverted microscopes to allow imaging of tissue • Relatively easy to set up and calibrate • Variants for living tissue and temperature dependent activation available 802D apparatus shown with 315C and custom transducer - Chandra lab WSU 802D - Permeabilized Fiber Apparatus
  20. 20. System Control & Software
  21. 21. • All experiments performed with our custom Real-Time Linux acquisition software (600A) and saved in an open format • Stretches, slacks and instantaneous tensile force all fully controllable and synchronized through software • Library of standard protocols allows for infinite customization • Straightforward to use despite Linux OS. Once sample attached and measured, load protocol and begin System Control & Software
  22. 22. Data Analysis • Camera based software suite for performing visualization and analysis • High frame rate CCD camera interfaces with control suite for SL control • Expansion edge detection and laser based modules in development 901D – High-Speed Video Sarcomere Length
  23. 23. What standard experiments can we perform? ✓ Passive & Active Stiffness ✓ Force - pCa ✓ kTR ✓ Length Dependent Activation ✓ Force - Velocity ✓ Slack Test ✓ And many more!
  24. 24. Determining single muscle fibre power using Temperature-jump and Force-control methods Tim West, PhD Structure & Motion Laboratory Royal Veterinary College Copyright 2019 Tim West and InsideScientific. All Rights Reserved.
  25. 25. Determining power in skeletal muscle fibres using Temperature-jump and Force-control approaches 2. The 600A software features that we use a. Force & SL control panel b. Protocol panel 3. Using the Force & SL Control Panel a. Between- and within-activation 1. Focus on force time-courses a. T-jump b. Force and Length-change during fibre shortening 4. Curve fitting to determine peak power a. ‘Normalised’ power (s-1) vs relative force (Fshort/Fisom) Aims: From Curtin et al. J Exp Biol 2015;218:2856-2863 From Wilson et al. Nature 2018; 554(7691):183-188 Stress(kPa)Stress(kPa) Wild rabbit; Extensor digiti V muscle Impala; Biceps femoris mussle
  26. 26. Temperature-jump using the Aurora 1400A permeabilized fibre apparatus Fibre bath 1 2 3 4 Relaxation 25 ◦C Activation 25 ◦C Activation 1 ◦C From Curtin et al. J Exp Biol 2015;218:2856-2863
  27. 27. Force, power and velocity data obtained from one rabbit ED-V fibre Curtin et al. J Exp Biol 2015;218:2856-2863
  28. 28. Comparison of skinned fibres and intact fibre bundles from wild rabbit peroneus longus and extensor digiti V Skinned fibres (N=141) Weighted mean±s.e.m. Intact fibre bundles (N=16) mean±s.e.m. P Isometric force, (kPa) 207.8±4.8 181.9±19.1 NS Normalized Max Power (s-1) 0.589±0.019 0.645±0.037 NS Rel force at max power 0.294±0.004 0.373±0.005 <0.001 V at max power (s-1) 2.012±0.065 1.61±0.088 <0.001 Max power (W litre-1) 122.6±4.6 121.3±16.1 NS Single skinned fibres produce the same power and force as intact fibre bundles from muscle of wild rabbits Curtin et al. J Exp Biol 2015;218:2856-2863
  29. 29. Q = Qmax ∙ Fo ∗ 𝐹 𝑄 𝑚𝑎𝑥 2 ∙ F ∙ 1 − F Fo ∗ 1 + F ∙ (Fo ∗ − 2 ∙ FQmax ൗ) FQma x 2 Fitting a power-force curve Qmax and FQmax and Fo ∗ are all free variables in the curve fitting procedure Q = normalized power (s-1) Qmax = maximum fitted power (s-1) F = normalized force (F/Fisometric) FQmax = Normalized force at Qmax Fo ∗ = The fitted force intercept (as shown) Qmax FQmax Fo ∗ F/Fisom Q=Power/FisomLo(s-1) Curtin et al. J Exp Biol 2015;218:2856-2863
  30. 30. Some local rules: Isometric Stress > 75 kPa Over the set (6+) activations using force control, isometric force remains ≥ 80% of the maximum observed Stress(kPa) Wild rabbit; extensor digit-V Disregard if period of force control shows: - Force oscillations - Force over- or under-shoot of target X 6
  31. 31. 0 5 1 0 1 5 2 0 2 5 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 mN Time (s) Activation 2 ◦C Activation 25 ◦C Relaxation 25 ◦C Fibre bath 1 2 3 4 Generating 4 separate force-control events during a single T-jump activation 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 Relative Force (F/Fisom) Power/FisomLo(s-1 )
  32. 32. The Force & SL Control window in the 600A software requires a measure of fibre stiffness 0 5000 10000 0.0 0.5 1.0 1.5 Time (ms) Force(mN) 5100 5110 5120 5130 5140 5150 0.6 0.8 1.0 1.2 0.990 0.995 1.000 1.005 1.010 Time (ms) Force(mN) RelativeLength -3.2 4.0 2.0 This Leopard fibre fragment: Biopsy of biceps femoris Fragment length = 0.64 mm = Lo Sarcomere length = 2.5 µm CSA (elliptical) = 0.0056 mm2 0.5% Lo = 3.2 mm 45.9
  33. 33. This Leopard fibre fragment: Biopsy of biceps femoris Fragment length = 0.64 mm Sarcomere length = 2.5 µm CSA (elliptical) = 0.0056 mm2 -3.2 4.0 2.0 0.85 45.9 0.30 0.30 0.32 0.32 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 Relative Force (F/Fisom) Power/FisomLo(s-1 ) 0 5 1 0 1 5 2 0 2 5 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 0 . 0 0 . 5 1 . 0 Fibre bath 1 2 3 4 The Force Controller Settings for generating force control measurements in pairs Force(mN) RelativeLength Time (s)
  34. 34. 0 5 1 0 1 5 2 0 2 5 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 0 . 0 0 . 5 1 . 0 9.00 9.01 9.02 9.03 0.0 0.5 1.0 9.2 0.6 0.8 1.0 12.00 12.01 12.02 12.03 0.0 0.5 1.0 12.2 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 Relative Force (F/Fisom) Power/FisomLo(s-1 ) Examples of stable force control and length change during a single T-jump activation Force(mN) RelativeLength Time (s) Time (s)Time (s) RelativeLength RelativeLength RelativeForce RelativeForce
  35. 35. 1000 Bath 1 0 s 51000 Data-Enable 52500 Length-Step 0.7 Lo 53000 Force-Sample 1 0 s 53500 Length-Step 1 Lo 54000 Bath 2 0 s 57000 SL-Trigger 0 s 58000 Bath 3 0 s 59900 Force-Sample 2 0 s 60000 Force-Step 0.35 d21 60020 Length-Step 0.7 Lo 60025 Force-Sample 3 0 s 60030 Length-Ramp1 Lo 5 ms 60900 Force-Sample 4 0 s 61000 Force-Step 0.3 d43 61020 Length-Step 0.7 Lo 61025 Force-Sample 5 0 s 61030 Length-Ramp1 Lo 5 ms 61900 Force-Sample 6 0 s 62000 Force-Step 0.25 d65 62020 Length-Step 0.7 Lo 62025 Force-Sample 7 0 s 62030 Length-Ramp1 Lo 5 ms 62900 Force-Sample 8 0 s 63000 Force-Step 0.2 d87 63020 Length-Step 0.7 Lo 63030 Length-Ramp1 Lo 5 ms 64000 Bath 4 0 s 67000 Length-Step 1 Lo 67500 Length-Step 0.7 Lo 68500 Length-Step 1 Lo 76000 Data-Disable 0 Stop Cold (1◦C) pre-activation and activation T-jump (25◦C) activation ‘force control’ events Relaxation at the test temperature (25◦C) 0 5 1 0 1 5 2 0 2 5 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 0 . 0 0 . 5 1 . 0 9.00 9.01 9.02 9.03 0.0 0.5 1.0 9.2 0.6 0.8 1.0 Time (s) RelativeLength RelativeForce Time (s) RelativeLength RelativeForce
  36. 36. 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 Relative Force (F/Fisom) Power/FisomLo(s-1 ) 0 5 1 0 1 5 2 0 2 5 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 mN Time (s) Same local rules apply: Over the 3 activations using force control, isometric force is at least 80% of the maximum Stress(kPa)Stress(kPa) Max Stress = 223 kPa Wild rabbit; extensor digit-V Impala; biceps femoris Leopard; biceps femoris 9.00 9.01 9.02 9.03 0.0 0.5 1.0 9.2 0.6 0.8 1.0 Time (s) RelativeLength RelativeForce Disregard if period of force control shows: - Force oscillations - Force over- or under-shoot of target Isometric Stress > 75 kPa
  37. 37. Volume, mm3 0.000 0.003 0.006 0.009 0.012 0.015 Power,W 0.0 0.5 1.0 1.5 2.0 cheetah Impala Lion Zebra • … • … • … • … From Wilson et al Nature 2018; 554(7691):183-188 High performance fibres from biceps femoris of lions and cheetahs are stronger, a little bit faster and more powerful than those from b. femoris of their prey species. …an example CSA, mm2 0.000 0.003 0.006 0.009 0.012 0.015 Force,mN 0.0 0.5 1.0 1.5 2.0 2.5 cheetah Impala Lion Zebra
  38. 38. Matt Borkowski Sales & Support Manager Aurora Scientific Tim West, PhD Chief Technician & Laboratory Manager Royal Veterinary College For additional information on the products and applications presented during this webinar, please contact the speakers below. Thank you!

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